Method and compositions for cellular immunotherapy

ABSTRACT

The present invention provides methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively transferring genetically modified tumor specific CD8+ T cells in the presence of tumor-specific, subset specific genetically modified CD4+ T cells, wherein the CD4+ T cells confer and/or augment a CD8+ T cells ability to sustain anti-tumor reactivity and increase and/or maximize tumor-specific proliferation of the tumor-specific CD8+ T cells of interest. Pharmaceutical formulations produced by the method, and methods of using the same, are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/006,641 filed May 5, 2014, now allowed, which is a U.S. nationalphase application of PCT/US2012/030388 filed Mar. 23, 2012, which claimsbenefit of U.S. Provisional Application No. 61/466,552 filed Mar. 23,2011 are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA018029 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to the field of biomedicine andspecifically methods useful for cancer therapy. In particular,embodiments of the invention relate to methods and compositions forcarrying out cellular immunotherapy.

BACKGROUND OF THE INVENTION

Studies in rodents have demonstrated that adoptive immunotherapy withantigen specific T cells is effective for cancer and infections, andthere is evidence this modality has therapeutic activity in humans¹⁻⁸.For clinical applications, it is necessary to isolate T cells of adesired antigen specificity or to engineer T cells to express receptorsthat target infected or transformed cells, and then expand these cellsin culture⁹⁻¹⁴. The transfer of T cell clones is appealing because itenables control of specificity and function, and facilitates evaluationof in vivo persistence, toxicity and efficacy. Additionally, in thesetting of allogeneic stem cell transplantation, the administration torecipients of T cell clones from the donor that target pathogens ormalignant cells can avoid graft-versus-host disease that occurs withinfusion of unselected donor T cells^(3,4,15). However, it is apparentfrom clinical studies that the efficacy of cultured T cells,particularly cloned CD8⁺ T cells, is frequently limited by their failureto persist after adoptive transfer^(16,17).

The pool of lymphocytes from which T cells for adoptive immunotherapycan be derived contains naïve and long-lived, antigen experienced memoryT cells (T_(M)). T_(M) can be divided further into subsets of centralmemory (T_(CM)) and effector memory (T_(EM)) cells that differ inphenotype, homing properties and function¹⁸. CD8⁺ T_(CM) express CD62Land CCR7 at the cell surface, which promote migration into lymph nodes,and proliferate rapidly if re-exposed to antigen. CD8⁺ T_(EM) lack cellsurface CD62L and preferentially migrate to peripheral tissues, andexhibit immediate effector function¹⁹. In response to antigenstimulation, CD8⁺ T_(CM) and T_(EM) both differentiate into cytolyticeffector T cells (T_(E)) that express a high level of granzymes andperforin, but are short-lived²⁰. Thus, the poor survival of T cells inclinical immunotherapy trials may simply result from theirdifferentiation during in vitro culture to T_(E) that are destined todie^(17,21,22). There is a need to identify cell populations and methodsthat provide enhanced survival of adoptively transferred T cells invivo.

SUMMARY

In one aspect, the present invention relates to methods and compositionsto confer and/or augment immune responses mediated by cellularimmunotherapy, such as by adoptively transferring tumor-specific, subsetspecific genetically modified CD4+ T cells, wherein the CD4+ T cellsconfer and/or augment the ability of CD8+ T cells to sustain anti-tumorreactivity and increase and/or maximize tumor-specific proliferation.

In one embodiment, the present invention provides a method of performingcellular immunotherapy in a subject having a disease or disorder byadministering to the subject a genetically modified cytotoxic Tlymphocyte cell preparation that provides a cellular immune response,wherein the cytotoxic T lymphocyte cell preparation comprises CD8+ Tcells that have a chimeric antigen receptor with an extracellularantibody variable domain specific for an antigen associated with thedisease or disorder and an intracellular signaling domain of a T cell orother receptors, such as co-stimulatory domains; and a geneticallymodified helper T lymphocyte cell preparation that exhibits apredominant Th1 phenotype as well as produce other cytokines, elicitsdirect tumor recognition and augments the genetically modified cytotoxicT lymphocyte cell preparations ability to mediate a cellular immuneresponse, wherein the helper T lymphocyte cell preparation comprisesCD4+ T cells that have a chimeric antigen receptor comprising anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor. Various modifications of the above methodare possible. For example, the chimeric antigen receptor modifying theCD4+ T cell and the CD8+ T cell can be the same or different. Inalternative embodiments, the T cells can be modified with a recombinantT cell receptor (TCR). TCR could be specific for any antigen, pathogenor tumor. There are TCRs for many tumor antigens in melanoma (MART1,gp100, for example), leukemia (WT1, minor histocompatibility antigens,for example), breast cancer (her2, NY-BR1, for example).

In another embodiment, the present invention provides an adoptivecellular immunotherapy composition having a genetically modified CD8+cytotoxic T lymphocyte cell preparation that elicits a cellular immuneresponse, wherein the cytotoxic T lymphocyte cell preparation comprisesCD8+ T cells that have a chimeric antigen receptor with an extracellularvariable domain antibody specific for an antigen associated with thedisease or disorder and an intracellular signaling domain of a T cell orother receptors, such as a costimulatory domain, and a geneticallymodified helper T lymphocyte cell preparation that exhibits apredominant Th1 phenotype as well as produce other cytokines, elicitsdirect tumor recognition and augments the ability of geneticallymodified cytotoxic T lymphocyte cell preparations to mediate a cellularimmune response, wherein the helper T lymphocyte cell preparation hasCD4+ T cells that have a chimeric antigen receptor with an extracellularantibody variable domain specific for the antigen associated with thedisease or disorder and an intracellular signaling domain of a T cellreceptor.

In yet another embodiment, the present invention provides an adoptivecellular immunotherapy composition having a chimeric antigen receptormodified tumor-specific CD8+ cytotoxic T lymphocyte cell preparationthat elicits a cellular immune response, wherein the cytotoxic Tlymphocyte cell preparation comprises CD8+ T cells that have a chimericantigen receptor comprising an extracellular single chain antibodyspecific for an antigen associated with the disease or disorder and anintracellular signaling domain of a T cell receptor, and anantigen-reactive chimeric antigen receptor modified naïve CD4+ T helpercell that is derived from CD45RO negative, CD62L positive CD4 positive Tcells, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides an adoptivecellular immunotherapy composition having an antigen specific CD8+cytotoxic T lymphocyte cell preparation that elicits a cellular immuneresponse comprising CD8+ T cells derived from the patient together withan antigen-reactive chimeric antigen receptor modified CD4+ T helpercell that elicits a Th1 cytokine response and augments the CD8+ immuneresponse to pathogens, wherein the helper T lymphocyte cell preparationwith CD4+ T cells that have a chimeric antigen receptor with anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor.

In another embodiment, the present invention provides an adoptivecellular immunotherapy composition with an antigen-reactive chimericantigen receptor modified CD4+ T helper cell that elicits direct tumorrecognition and augments the CD8+ immune response to pathogens, whereinthe helper T lymphocyte cell preparation comprises CD4+ T cells thathave a chimeric antigen receptor comprising an extracellular antibodyvariable domain specific for an antigen associated with a disease ordisorder and an intracellular signaling domain of a T cell receptor.

In another aspect, the present invention provides a method ofmanufacturing an adoptive immunotherapy composition by obtaining achimeric antigen receptor modified tumor-specific CD8+ cytotoxic Tlymphocyte cell preparation that elicits a cellular immune response andan antigen-reactive chimeric antigen receptor, wherein the modifiedcytotoxic T lymphocyte cell preparation comprises CD8+ T cells that havea chimeric antigen receptor with an extracellular antibody variabledomain specific for an antigen associated with the disease or disorderand an intracellular signaling module of a T cell receptor; andobtaining a modified naïve CD4+ T helper cell that elicits a Th1cytokine response, wherein the modified helper T lymphocyte cellpreparation comprises CD4+ cells that have a chimeric antigen receptorwith an extracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor.

In another embodiment, the present invention provides a method ofmanufacturing an adoptive immunotherapy composition by obtaining amodified naïve CD4+ T helper cell that elicits a Th1 cytokine response,wherein the modified helper T lymphocyte cell preparation comprises CD4+T cells that have a chimeric antigen receptor comprising anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor, and combining the modified naïve CD4+ Thelper cell with an antigen specific central memory CD8+ cytotoxic Tlymphocyte cell preparation that has a chimeric antigen receptor with anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell or other receptors.

In one embodiment, the present invention provides a method of performingcellular immunotherapy in subject having a disease or disorder byadministering to the subject a genetically modified helper T lymphocytecell preparation, wherein the modified helper T lymphocyte cellpreparation comprises CD4+ T cells that have a chimeric antigen receptorcomprising an extracellular antibody variable domain specific for anantigen associated with the disease or disorder and an intracellularsignaling module of a T cell receptor.

These and other embodiments of the invention are described further inthe accompanying specification, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the phenotype and analysis of chimeric antigen receptor(CAR) expression in a CAR-transduced with ROR1-CAR encoding lentivirus,and an untransduced CD8+ T cell line as a control. The ROR1-CAR cassettecontains a truncated EGFR that serves as transduction marker and can bedetected by staining with anti-EGFR monoclonal antibodies. TruncatedFc-ROR1 fusion protein binds directly to the antigen-binding domain ofthe ROR1-CAR and selectively stains the ROR1-CAR transduced but not theuntransduced control T cell line. Expression of the ROR1-CAR on the cellsurface of CD8+ T cells is measured directly by binding to ROR1-Fcfusion protein and indirectly by expression of a truncated EGFR that isencoded downstream of a 2A sequence in the vector.

FIG. 2 shows cytolytic activity of CD8+ T cells expressing aROR1-specific chimeric antigen receptor against a panel of humanROR1-positive tumor cell lines (K562) and primary tumor cells (B-CLL)and autologous normal B-cells in a ⁵¹Cr release assay. Consistent withthe uniform expression of ROR1 on malignant but not on mature normal Bcells, genetically modified CD8+ ROR1-CAR T cells only lysed ROR1+ tumorcells but not mature normal B cells. CD8+ ROR1-CAR T cells exertspecific lytic activity against ROR1-positive tumor cells includingprimary CLL, but not against normal B cells.

FIG. 3 shows the phenotype and CAR expression of a ROR1-CAR transducedand an untransduced CD4+ T cell line as a control. Expression of theROR1-CAR on the cell surface of CD4+ T cells is measured by specificbinding to ROR1-Fc fusion protein. Truncated Fc ROR1 fusion protein butnot Fc protein alone binds directly to the ROR1-CAR and selectivelystains the ROR1-CAR transduced but not the untransduced control CD4+ Tcell line confirming expression of the ROR1-CAR on the cell surface andbinding to ROR1-protein. Expression of the ROR1-CAR on the cell surfaceof CD4+ T cells is measured by specific binding to ROR1-Fc fusionprotein, but not to a control Fc fusion protein.

FIGS. 4A and 4B show weak but specific cytolytic activity of CD4+ROR1-CAR T cells in a ⁵¹Cr release assay against a panel ofROR1-positive tumor cells including primary CLL, the mantle celllymphoma line Jeko-1, K562 cells that were stably transfected with ROR1(K562/ROR1), but not native ROR1-negative K562 cells. CD4+ ROR1-CAR Tcells exert weak but specific lytic activity against ROR1-positive tumorcells.

FIGS. 5A and 5B show the results from an IFNγ ELISA (FIG. 5A) andmultiplex cytokine assay (FIG. 5B). Cytokine secretion of CD4+ and CD8+ROR1-CAR T cell lines. CD4+ROR1-CAR and CD8 ROR1-CAR T cells wereco-incubated with ROR1+ tumor cells, and levels of interferon gamma(IFNγ) was measured by ELISA (5A), and IFNγ, TNFα, IL-2, IL-4, IL-10 andIL-17 were measured by Luminex assay (5B). CD4+ ROR1-CAR modified Tcells specifically recognize ROR1-positive tumor cells and tumor celllines and produce higher amounts of Th1 cytokines including IFN-γ, TNF-αand particularly IL-2 than CD8+ ROR1-CAR modified T cells. These datademonstrate that CD4+ ROR1-CAR T cells exert helper effector functionsafter stimulation through the ROR1-CAR and in addition to mediatingdirect anti-tumor reactivity, could also be utilized to augment theability of CD8+ ROR1-CAR modified T cells to mediate a cellular immuneresponse.

FIG. 6 depicts the results of a proliferation study showing that CD4+ROR1-CAR T cells are induced to proliferate after stimulation withROR1-positive tumor cell lines and primary tumor cells (CFSE assay) andthat both the percentage of proliferating cells and number of celldivisions that the proliferating subset underwent were significantlyhigher compared to CD8+ ROR1-CAR modified T cells. CD4+ ROR1-CAR T cellsproliferate more vigorously after stimulation with ROR1-positive tumorcells (K562/ROR1, primary CLL, and Jeko MCL) compared to CD8+ ROR1-CARCTLs.

FIG. 7 shows polyclonal unselected CD4+ ROR1 CAR T cells provide help toCD8+ ROR1-CAR CTLs by promoting their proliferation in response totumor. CD4+ ROR1-CAR T cells (derived from bulk CD4+ T cells)significantly increased proliferation of polyclonal unselected CD8+ROR1-CAR CTLs (18% in individual culture→31.5% after co-culture withCD4+ CAR T cells).

FIGS. 8A-8D show the generation of CD4+ CAR T cell lines from flow sortpurified CD4+ naïve, central memory and effector memory subsets andanalysis of T-cell function. Cytokine profile and proliferative capacitysuggest that CD4+ ROR1-CAR T cells derived from naïve CD4+ T cells maybe best suited to provide help to CD8+ CTLs. Similar data were obtainedin experiments comparing the function of CD4+ CAR T-cell linesexpressing a CD19-specific CAR. FIG. 8A shows flow sort purification ofnaïve, central and effector memory CD4+ T cells based on expression ofCD45RA, CD45RO, CD62L. FIG. 8B shows analysis of proliferation ofROR1-CAR T cell lines that were derived by lentiviral transduction ofsort purified naïve, central and effector memory CD4+ T cells (CFSEassay). FIG. 8C shows analysis of cytokine secretion of ROR1-CAR T celllines from sort purified naïve, central and effector memory CD4+ T cells(Luminex assay). FIG. 8D shows analysis of cytokine secretion ofCD19-CAR T-cell lines from sort purified naïve, central and effectormemory CD4+ T cells (Luminex assay). The cytokine profile obtained bymultiplex cytokine analysis (FIG. 8B) and proliferative capacity by CFSEstaining (FIG. 8C) shows that CD4+ ROR1-CAR modified T cells derivedfrom the naïve subset produced the highest levels of Th1 cytokines andproliferated most vigorously after stimulation with ROR1-positive tumorcells, suggesting they may be best suited to augment CD8+ ROR1-CAR CTLs.Analysis of cytokine secretion of CD19-CAR T cell lines fromsort-purified naïve, central and effector memory CD4+ T cells (Luminexassay), demonstrates that the activity of CD4 T cell subsets isgeneralizable to many CARs.

FIG. 9 shows co-culture of CD8+ ROR1-CAR modified T cells with CD4+ROR1-CAR modified T cells (but not untransduced control CD4+ T cells).Co-culture of CD8+ ROR1-CAR CTLs and CD4+ ROR1-CAR T cell lines derivedfrom naïve, central and effector memory subsets to define the optimalcombination of CD8+ and CD4+ T cells that would allow maximumproliferation of CD8+ ROR1-CAR CTLs. CD4 naïve ROR1-CAR T cells providethe greatest proliferation of CD8 central memory ROR1-CAR CTLs. Coculture leads to an increase in tumor-specific proliferation of the CD8+subset, and that maximum proliferation of the CD8+ subset is observedafter co-culture with CD4+ ROR1-CAR T cells derived from naïve CD4+ Tcells.

FIG. 10 shows the superior ability of CD4+ CAR T-cell lines derived fromthe naïve subset to augment tumor-specific proliferation of centralmemory-derived CD8+ CAR CTL in co-culture experiments with CD8+ CD19-CARCTLs and CD4+ CD19-CAR T-cell lines, stimulated with the CD19+ mantlecell lymphoma tumor line Jeko-1. The superior ability of CD4+ CAR T-celllines derived from the naïve subset to augment tumor-specificproliferation of central memory-derived CD8+ CAR CTL was confirmed inco-culture experiments with CD8+ CD19-CAR CTLs and CD4+ CD19-CAR T-celllines, stimulated with the CD19+ mantle cell lymphoma tumor line Jeko-1.

FIGS. 11A and 11B show that CD8+ CAR T cells and CD4+ CAR T cellsindependently confer direct anti-tumor efficacy in a lymphoma model inimmunodeficient mice (NOD/SCID-Raji). Groups of mice (n=3) wereinoculated with firefly-luciferase expressing Raji tumor cells via tailvein injection and treated with a single dose of 10×10̂6 T cells. Micereceived either CD19-CAR transduced or control mock-transduced CD8+central memory-derived (FIG. 11A), or CD19-CAR transduced or controlmock-transduced CD4+ naïve-derived T cells (FIG. 11B). Tumor burden anddistribution was analyzed using serial bioluminescence imaging.

FIG. 12 shows the augmentation and synergistic effect CD4+ ROR1-CARmodified T cells on the anti-tumor efficacy of CD8+ ROR1-CAR CTLs in amouse tumor model of systemic mantle cell lymphoma (NSG/Jeko-1-ffLuc).Anti-tumor efficacy of ROR1-CAR modified CD8+ and CD4+ T cells in amouse tumor model of systemic aggressive mantle cell lymphoma(NSG/Jeko-1). Analysis of tumor burden using bioluminescence imagingafter adoptive transfer of CD8+ ROR1-CAR CTLs, CD4+ ROR1-CAR T cells ora combination of CD8+ and CD4+ ROR1-CAR T cells T cells. All micereceived the same total dose of CAR T cells.

FIGS. 13A-13D show synergy of CD8+ and CD4+ CD19-CAR T cells in a mousemodel of systemic lymphoma (NSG/Raji). NSG mice were inoculated withfirefly-luciferase transduced Raji tumor cells. Engraftment of the Rajitumor was confirmed by bioluminescence imaging on day 6 after tumorinoculation (before treatment) (treatment scheme shown in FIG. 13A,tumor engraftment by bioluminescence shown in FIG. 13B. Groups of mice(n=5) were then treated with either CD8+ CD19-CAR modified T cells, or acombined T-cell product that contained both CD8+ and CD4+ CD19-CAR Tcells. All mice received the same total dose of T cells (10×10⁶).Analysis of tumor burden using bioluminescence imaging showed completeeradication of the Raji tumors in the cohorts of mice treated with CD8+CD19-CAR T cells, and in mice treated with the combined CD8+ and CD4+CD19-CAR T-cell product (after treatment middle black and grey bars)(FIG. 13B). The mice were then challenged with a second inoculum of Rajitumor cells and the frequency of CD4+ and CD8+ CAR T cells in theperipheral blood, and tumor engraftment were analyzed. In mice treatedwith a combined CD8+ and CD4+ CAR T-cell product, significantly higherlevels CD8+ CAR T cells after the tumor challenge (FIG. 13C, lowerpanels), and complete rejection of the Raji inoculum (after tumorchallenge right grey bar, FIG. 13B). In contrast, in mice that hadreceived CD8+ CD19-CAR CTL alone, we did not detect an increase in CAR Tcells after the tumor challenge (FIG. 13C) and the Raji tumor cells wereable to engraft (after tumor challenge right black bar, panel FIG. 13B).

DETAILED DESCRIPTION

“T cells” or “T lymphocytes” as used herein may be from any mammalian,preferably primate, species, including monkeys, dogs, and humans. Insome embodiments the T cells are allogeneic (from the same species butdifferent donor) as the recipient subject; in some embodiments the Tcells are autologous (the donor and the recipient are the same); in someembodiments the T cells arc syngeneic (the donor and the recipients aredifferent but are identical twins).

Cytotoxic T lymphocyte (CTL) as used herein refers to a T lymphocytethat expresses CD8 on the surface thereof (i.e., a CD8⁺ T cell). In someembodiments such cells are preferably “memory” T cells (T_(M) cells)that are antigen-experienced.

“Central memory” T cell (or “T_(CM)”) as used herein refers to anantigen experienced CTL that expresses CD62L and CD45RO on the surfacethereof, and does not express or has decreased expression of CD45RA ascompared to naïve cells. In embodiments, central memory cells arepositive for expression CD62L, CCR7, CD28, CD127, CD45RO, and CD95, andhave decreased expression of CD54RA as compared to naïve cells.

“Effector memory” T cell (or “T_(EM)”) as used herein refers to anantigen experienced CTL that does not express or has decreasedexpression of CD62L on the surface thereof as compared to central memorycells, and does not express or has decreased expression of CD45RA ascompared to naïve cell. In embodiments, effector memory cells arenegative for expression CD62L, CCR7, CD28, CD45RA, and are positive forCD127 as compared to naïve cells or central memory cells.

“Naïve” T cells as used herein refers to a non-antigen experienced Tlymphocyte that expresses CD62L and CD45RA, and does not express or hasdecreased expression of CD45RO− as compared to central memory cells. Insome embodiments, naïve CD8+ T lymphocytes are characterized by theexpression of phenotypic markers of naïve T cells including CD62L, CCR7,CD28, CD3, CD127, and CD45RA.

“Effector” or “T_(E)” T cells as used herein refers to a antigenexperienced cytotoxic T lymphocyte cells that do not express or havedecreased expression of CD62L, CCR7, CD28, and are positive for granzymeB and perforin as compared to central memory cells.

“Enriched” and “depleted” as used herein to describe amounts of celltypes in a mixture refers to the subjecting of the mixture of the cellsto a process or step which results in an increase in the number of the“enriched” type and a decrease in the number of the “depleted” cells.Thus, depending upon the source of the original population of cellssubjected to the enriching process, a mixture or composition may contain60, 70, 80, 90, 95, or 99 percent or more (in number or count) of the“enriched” cells and 40, 30, 20, 10, 5 or 1 percent or less (in numberor count) of the “depleted” cells.

Interleukin-15 is a known and described in, for example, U.S. Pat. No.6,344,192.

“CAR” as used herein refers to chimeric antigen receptor comprising anextracellular variable domain of an antibody specific for an antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell or other receptors, such as a costimulatory domain.

Modes of the Disclosure

CD4+ T lymphocytes during in vitro culture significantly increaseproliferation, persistence and anti-tumor reactivity of tumor-specificCD8+ T cells in vitro and in vivo. In some embodiments, naïve CD4+ Tcells possess an intrinsic programming that leads to superior helperactivity compared to CD4+ T cells derived from central and effectormemory, or bulk CD4+ T cells.

In embodiments, tumor-reactive CD4+ T cells are modified with asingle-chain antibody-derived chimeric antigen receptor (CAR) specificfor the orphan tyrosine kinase receptor ROR1 or for the CD19 molecule.ROR1 is uniformly expressed on chronic lymphocytic leukemia (CLL) andmantle cell lymphoma (MCL) and ROR1-specific CAR from an anti-ROR1monoclonal antibody (mAb) confers specific recognition of malignant, butnot mature normal B-cells when expressed in CD8+ cytotoxic T cells(CTLs). ROR1-CAR T cells from bulk and flow sort purified naïve, centraland effector memory CD4+ T cells are obtained from the peripheral bloodof both healthy donors and CLL patients. CD4+ CAR T cells had specificbut weak cytolytic activity against ROR1+ tumors including primary CLL,the MCL line Jeko-1, and K562 cells transfected with ROR1. Multiplexcytokine analysis detects high-level production of Th1 cytokines withsignificantly higher levels of IFNγ, TNFα, and particularly IL-2compared to CD8+ CAR CTLs. CFSE staining shows dramatically higherproliferation after stimulation with ROR1-positive tumor cells, withboth the percentage of cells that were induced to proliferate and thenumber of cell divisions that the proliferating subset underwent beingsignificantly higher compared to CD8+ CAR CTL. CD4+ T cells obtainedfrom both healthy donors and CLL patients acquire anti-tumor reactivityafter genetic modification with a ROR1-specific CAR. Moreover, theability to proliferate in the absence of exogenous cytokines and toproduce high levels of Th1 cytokines demonstrates that CD4+ CAR T cellsexert typical helper functions after stimulation through the CAR andsuggests that in addition to conferring direct anti-tumor effects, couldbe utilized to augment tumor-specific CD8+ CTL.

The cytokine profile and proliferative capacity of ROR1-CAR T cellsderived from flow sort purified CD4+ naïve, central and effector memorysubsets is obtained. The CD4+ CAR T cells, derived from the naïveCD45RA+ CD45RO− CD62L+ subset, produces the highest levels of Th1cytokines, especially IL-2, and proliferates in response to ROR1+ tumorcells. Indeed, in co-culture experiments, the addition ofCAR-transduced, but not untransduced CD4+ T cells leads to a significantincrease in tumor-specific proliferation of CD8+ CAR CTLs. In someembodiments, CAR-modified CD4+T cells derived from naïve rather thancentral and effector memory subsets or bulk CD4+ T cells results inenhanced proliferation of CD8+ CAR CTL.

CD8+ central memory T cells have an intrinsic programming that allowsthem to persist for extended periods after administration, which makesthem the preferred subset of CD8+ T cells for immunotherapy. Inembodiments, ROR1-CAR or CD19 CAR modified CTLs from sort purified CD8+central memory T cells and CD4+ naïve CAR-modified T cells provideenhanced proliferation of the CD8+ T cell subset. In embodiments,tumor-specific CD4+ T cells exert anti-tumor reactivity and provide helpto tumor-specific CD8+ T cells in vitro and in vivo. In a specificembodiment, tumor-specific CD4+ T cells from the naïve subset areutilized.

In another embodiment, the CD8+ and CD4+ T cells can be modified with aT cell receptor (TCR). The TCR could be specific for any antigen,pathogen or tumor (there are TCRs for many tumor antigens in melanoma(MART1, gp100 for example), leukemia (WT1, minor histocompatibilityantigens for example), breast cancer (her2, NY-BR1 for example).

Compositions

The disclosure provides for an adoptive cellular immunotherapycomposition comprising a genetically modified helper T lymphocyte cellpreparation that augments the genetically modified cytotoxic Tlymphocyte cell preparations ability to mediate a cellular immuneresponse, wherein the helper T lymphocyte cell preparation comprisesCD4+ T cells that have a chimeric antigen receptor comprising anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor or other receptors.

In some embodiments, an adoptive cellular immunotherapy compositionfurther comprises a chimeric antigen receptor modified tumor-specificCD8+ cytotoxic T lymphocyte cell preparation that elicits a cellularimmune response, wherein the cytotoxic T lymphocyte cell preparationcomprises CD8+ T cells that have a chimeric antigen receptor comprisingan extracellular single chain antibody specific for an antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor.

In some embodiments, an adoptive cellular immunotherapy compositioncomprises a chimeric antigen receptor modified tumor-specific CD8+cytotoxic T lymphocyte cell preparation that elicits a cellular immuneresponse, wherein the cytotoxic T lymphocyte cell preparation comprisesCD8+ T cells that have a chimeric antigen receptor comprising anextracellular single chain antibody specific for an antigen associatedwith the disease or disorder and an intracellular signaling domain of aT cell receptor, in combination with an antigen-reactive chimericantigen receptor modified naïve CD4+ T helper cell derived from CD45ROnegative, CD62L positive CD4 positive T cells, and a pharmaceuticallyacceptable carrier.

In other embodiments, an adoptive cellular immunotherapy compositioncomprises an antigen specific CD8+ cytotoxic T lymphocyte cellpreparation that elicits a cellular immune response derived from thepatient combined with an antigen-reactive chimeric antigen receptormodified naïve CD4+ T helper cell that augments the CD8+ immuneresponse, wherein the helper T lymphocyte cell preparation comprisesCD4+ T cells that have a chimeric antigen receptor comprising anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor.

In a further embodiment, an adoptive cellular immunotherapy compositioncomprises an antigen-reactive chimeric antigen receptor modified naïveCD4+ T helper cell that augments the CD8+ immune response, wherein thehelper T lymphocyte cell preparation comprises CD4+ T cells that have achimeric antigen receptor comprising an extracellular antibody variabledomain specific for an antigen associated with a disease or disorder andan intracellular signaling domain of a T cell receptor.

In embodiments, the CD4+ T helper lymphocyte cell is selected from thegroup consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, or bulk CD4+ T cells. In some embodiments,CD4+ helper lymphocyte cell is a naïve CD4+ T cell, wherein the naïveCD4+ T cell comprises a CD45RO−, CD45RA+, CD62L+ CD4+ T cell. Inembodiments, the CD8+ T cytotoxic lymphocyte cell is selected from thegroup consisting of naïve CD8+ T cells, central memory CD8+ T cells,effector memory CD8+ T cells or bulk CD8+ T cells. In some embodiments,the CD8+ cytotoxic T lymphocyte cell is a central memory T cell whereinthe central memory T cell comprises a CD45RO+, CD62L+, CD8+ T cell. Inyet other embodiments, the CD8+ cytotoxic T lymphocyte cell is a centralmemory T cell and the CD4+ helper T lymphocyte cell is a naïve CD4+ Tcell.

In alternative embodiments, the T cells can be modified with arecombinant T cell receptor. TCR could be specific for any antigen,pathogen or tumor. There are TCRs for many tumor antigens in melanoma(MART1, gp100, for example), leukemia (WT1, minor histocompatibilityantigens, for example), breast cancer (her2, NY-BR1, for example).

Selection and Sorting of T Lymphocyte Populations

The compositions described herein provide for antigen reactive CD4+ andCD8+ T lymphocytes.

T lymphocytes can be collected in accordance with known techniques andenriched or depleted by known techniques such as affinity binding toantibodies such as flow cytometry and/or immunomagnetic selection. Afterenrichment and/or depletion steps, in vitro expansion of the desired Tlymphocytes can be carried out in accordance with known techniques(including but not limited to those described in U.S. Pat. No. 6,040,177to Riddell et al.), or variations thereof that will be apparent to thoseskilled in the art.

For example, the desired T cell population or subpopulation may beexpanded by adding an initial T lymphocyte population to a culturemedium in vitro, and then adding to the culture medium feeder cells,such as non-dividing peripheral blood mononuclear cells (PBMC) (e.g.,such that the resulting population of cells contains at least about 5,10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in theinitial population to be expanded); and incubating the culture (e.g.,for a time sufficient to expand the numbers of T cells). Thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads. The order of addition of the Tcells and feeder cells to the culture media can be reversed if desired.The culture can typically be incubated under conditions of temperatureand the like that are suitable for the growth of T lymphocytes. For thegrowth of human T lymphocytes, for example, the temperature willgenerally be at least about 25 degrees Celsius, preferably at leastabout 30 degrees, more preferably about 37 degrees.

The T lymphocytes expanded include cytotoxic T lymphocytes (CTL) andhelper T lymphocytes that are specific for an antigen present on a humantumor or a pathogen.

Optionally, the expansion method may further comprise the step of addingnon-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.LCL can be irradiated with gamma rays in the range of about 6000 to10,000 rads. The LCL feeder cells may be provided in any suitableamount, such as a ratio of LCL feeder cells to initial T lymphocytes ofat least about 10:1.

Optionally, the expansion method may further comprise the step of addinganti-CD3 monoclonal antibody to the culture medium (e.g., at aconcentration of at least about 0.5 ng/ml). Optionally, the expansionmethod may further comprise the step of adding IL-2 and/or IL-15 to theculture medium (e.g., wherein the concentration of IL-2 is at leastabout 10 units/ml).

After isolation of T lymphocytes both cytotoxic and helper T lymphocytescan be sorted into naïve, memory, and effector T cell subpopulationseither before or after expansion.

CD8+ cells can be obtained by using standard methods. In someembodiments, CD8+ cells are further sorted into naïve, central memory,and effector cells by identifying cell surface antigens that areassociated with each of those types of CD8+ cells. In embodiments,memory T cells are present in both CD62L+ and CD62L− subsets of CD8+peripheral blood lymphocytes. PBMC are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies.In some embodiments, the expression of phenotypic markers of centralmemory T_(CM) include CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and arenegative for granzyme B. In some embodiments, central memory T cells areCD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T_(E) arenegative for CD62L, CCR7, CD28, and CD127, and positive for granzyme Band perforin. In some embodiments, naïve CD8+ T lymphocytes arecharacterized by the expression of phenotypic markers of naïve T cellsincluding CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

Whether a cell or cell population is positive for a particular cellsurface marker can be determined by flow cytometry using staining with aspecific antibody for the surface marker and an isotype matched controlantibody. A cell population negative for a marker refers to the absenceof significant staining of the cell population with the specificantibody above the isotype control, positive refers to uniform stainingof the cell population above the isotype control. In some embodiments, adecrease in expression of one or markers refers to loss of 1 log 10 inthe mean fluorescence intensity and/or decrease of percentage of cellsthat exhibit the marker of at least 20% of the cells, 25% of-the cells,30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells,50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells,70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells,90% of the cell, 95% of the cells, and 100% of the cells and any %between 20 and 100% when compared to a reference cell population. Insome embodiments, a cell population positive for of one or markersrefers to a percentage of cells that exhibit the marker of at least 50%of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70%of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90%of the cell, 95% of the cells, and 100% of the cells and any % between50 and 100% when compared to a reference cell population.

CD4+ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naïve CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+ CD4+T cell. In some embodiments, central memory CD4+ cells are CD62Lpositive and CD45RO positive. In some embodiments, effector CD4+ cellsare CD62L and CD45RO negative.

Populations of CD4+ and CD8+ that are antigen specific can be obtainedby stimulating naïve or antigen specific T lymphocytes with antigen. Forexample, antigen specific T cell clones can be generated toCytomegalovirus antigens by isolating T cells from infected subjects andstimulating the cells in vitro with the same antigen. Naïve T cells mayalso be used. Any number of antigens from tumor cells, cancer cells, orinfectious agents may be utilized. Examples of such antigens include HIVantigens, HCV antigens, HBV antigens, CMV antigens, parasitic antigens,and tumor antigens such as orphan tyrosine kinase receptor ROR1, tEGFR,Her2, L1-CAM, CD19, CD20, CD22, mesothelin, and CEA. In someembodiments, the adoptive cellular immunotherapy compositions are usefulin the treatment of a disease or disorder including a solid tumor,hematologic malignancy, melanoma, or infection with a virus.

Modification of T Lymphocyte Populations

In some embodiments it may be desired to introduce functional genes intothe T cells to be used in immunotherapy in accordance with the presentdisclosure. For example, the introduced gene or genes may improve theefficacy of therapy by promoting the viability and/or function oftransferred T cells; or they may provide a genetic marker to permitselection and/or evaluation of in vivo survival or migration; or theymay incorporate functions that improve the safety of immunotherapy, forexample, by making the cell susceptible to negative selection in vivo asdescribed by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); andRiddell et al., Human Gene Therapy 3:319-338 (1992); see also thepublications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al.describing the use of bifunctional selectable fusion genes derived fromfusing a dominant positive selectable marker with a negative selectablemarker. This can be carried out in accordance with known techniques(see, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at columns 14-17)or variations thereof that will be apparent to those skilled in the artbased upon the present disclosure.

In embodiments, T cells are modified with chimeric antigen receptors(CAR). In some embodiments, CARs comprise a single-chain antibodyfragment (scFv) that is derived from the variable heavy (VH) andvariable light (VL) chains of a monoclonal antibody (mAb) linked to theTCR CD3+ chain that mediates T-cell activation and cytotoxicity.Costimulatory signals can also be provided through the CAR by fusing thecostimulatory domain of CD28 or 4-1BB to the CD3+ chain. CARs arespecific for cell surface molecules independent from HLA, thusovercoming the limitations of TCR-recognition including HLA-restrictionand low levels of HLA-expression on tumor cells.

CARs can be constructed with a specificity for any cell surface markerby utilizing antigen binding fragments or antibody variable domains of,for example, antibody molecules. The antigen binding molecules can belinked to one or more cell signaling modules. In embodiments, cellsignaling modules include CD3 transmembrane domain, CD3 intracellularsignaling domains, and CD 28 transmembrane domains. In embodiments, theintracellular signaling domain comprises a CD28 transmembrane andsignaling domain linked to a CD3 intracellular domain. In someembodiments, a CAR can also include a transduction marker, such astEGFR.

In embodiments, the intracellular signaling domain of the CD8+ cytotoxicT cells is the same as the intracellular signaling domain of the CD4+helper T cells. In other embodiments, the intracellular signaling domainof the CD8+ cytotoxic T cells is different than the intracellularsignaling domain of the CD4+ helper T cells.

In some embodiments, the CD8+ T cell and the CD4+ T cell are bothgenetically modified with an antibody heavy chain domain thatspecifically binds a pathogen-specific cell surface antigen. Inembodiments, CARs are specific for cell surface expressed antigensassociated with pathogens, tumors, or cancer cells. In some embodiments,a CAR is specific for HIV antigens, HCV antigens, HBV antigens, CMVantigens, parasitic antigens, and tumor antigens such as orphan tyrosinekinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin,and CEA. Methods for producing a CAR are described herein and can alsobe found in U.S. Pat. No. 6,410,319 by Forman and WO 2002/077029, U.S.Pat. No. 7,446,191, 2010/065818, 2010/025177, 2007/059298, and U.S. Pat.No. 7,514,537 by Jensen et al. and as described by Berger C. et al., J.Clinical Investigation, 118:1 294-308 (2008), which are herebyincorporated by reference.

In embodiments, the same or a different CAR can be introduced into eachof CD4+ and CD8+ T lymphocytes. In embodiments, the CAR in each of thesepopulations has an antigen binding molecule that specifically binds tothe same antigen. The cellular signaling modules can differ. Inembodiments each of the CD4 or CD8 T lymphocytes can be sorted in tonaïve, central memory, effector memory or effector cells prior totransduction. In alternative embodiments, each of the CD4 or CD8 Tlymphocytes can be sorted in to naïve, central memory, effector memory,or effector cells prior to transduction.

In alternative embodiments, the T cells can be modified with arecombinant T cell receptor. TCR could be specific for any antigen,pathogen or tumor. There are TCRs for many tumor antigens in melanoma(MART1, gp100 for example), leukemia (WT1, minor histocompatibilityantigens for example), breast cancer (her2, NY-BR1 for example).

Various infection techniques have been developed which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a currently preferred approach to the transduction of Tlymphocytes of the present invention. The viral vectors which have beenused in this way include virus vectors derived from simian virus 40,adenoviruses, adeno-associated virus (AAV), lentiviral vectors, andretroviruses. Thus, gene transfer and expression methods are numerousbut essentially function to introduce and express genetic material inmammalian cells. Several of the above techniques have been used totransduce hematopoietic or lymphoid cells, including calcium phosphatetransfection, protoplast fusion, electroporation, and infection withrecombinant adenovirus, adeno-associated virus and retrovirus vectors.Primary T lymphocytes have been successfully transduced byelectroporation and by retroviral infection.

Retroviral vectors provide a highly efficient method for gene transferinto eukaryotic cells. Moreover, retroviral integration takes place in acontrolled fashion and results in the stable integration of one or a fewcopies of the new genetic information per cell.

It is contemplated that overexpression of a stimulatory factor (forexample, a lymphokine or a cytokine) may be toxic to the treatedindividual. Therefore, it is within the scope of the invention toinclude gene segments that cause the T cells of the invention to besusceptible to negative selection in vivo. By “negative selection” ismeant that the infused cell can be eliminated as a result of a change inthe in vivo condition of the individual. The negative selectablephenotype may result from the insertion of a gene that conferssensitivity to an administered agent, for example, a compound. Negativeselectable genes are known in the art, and include, inter alia thefollowing: the Herpes simplex virus type I thymidine kinase (HSV-I TK)gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovirsensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT)gene, the cellular adenine phosphoribosyltransferase (APRT) gene,bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci.USA. 89:33, 1992).

In some embodiments it may be useful to include in the T cells apositive marker that enables the selection of cells of the negativeselectable phenotype in vitro. The positive selectable marker may be agene which, upon being introduced into the host cell expresses adominant phenotype permitting positive selection of cells carrying thegene. Genes of this type are known in the art, and include, inter alia,hygromycin-B phosphotransferase gene (hph) which confers resistance tohygromycin B, the amino glycoside phosphotransferase gene (neo or aph)from Tn5 which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, the adenosine daminase gene (ADA),and the multi-drug resistance (MDR) gene.

Preferably, the positive selectable marker and the negative selectableelement are linked such that loss of the negative selectable elementnecessarily also is accompanied by loss of the positive selectablemarker. Even more preferably, the positive and negative selectablemarkers are fused so that loss of one obligatorily leads to loss of theother. An example of a fused polynucleotide that yields as an expressionproduct a polypeptide that confers both the desired positive andnegative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See Lupton et al., Mol. Cell. Biol. 11:3374-3378,1991. In addition, in preferred embodiments, the polynucleotides of theinvention encoding the chimeric receptors are in retroviral vectorscontaining the fused gene, particularly those that confer hygromycin Bresistance for positive selection in vitro, and ganciclovir sensitivityfor negative selection in vivo, for example the HyTK retroviral vectordescribed in Lupton, S. D. et al. (1991), supra. See also thepublications of PCT/US91/08442 and PCT/US94/05601, by S. D. Lupton,describing the use of bifunctional selectable fusion genes derived fromfusing a dominant positive selectable marker with negative selectablemarkers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, nco, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Especially preferred markers are bifunctional selectable fusion geneswherein the positive selectable marker is derived from hph or neo, andthe negative selectable marker is derived from cytosine deaminase or aTK gene or selectable marker.

A variety of methods can be employed for transducing T lymphocytes, asis well known in the art. For example, retroviral transductions can becarried out as follows: on day 1 after stimulation using REM asdescribed herein, provide the cells with 20-30 units/ml IL-2; on day 3,replace one half of the medium with retroviral supernatant preparedaccording to standard methods and then supplement the cultures with 5μg/ml polybrene and 20-30 units/ml IL-2; on day 4, wash the cells andplace them in fresh culture medium supplemented with 20-30 units/mlIL-2; on day 5, repeat the exposure to retrovirus; on day 6, place thecells in selective medium (containing, e.g., an antibiotic correspondingto an antibiotic resistance gene provided in the retroviral vector)supplemented with 30 units/ml IL-2; on day 13, separate viable cellsfrom dead cells using Ficoll Hypaque density gradient separation andthen subclone the viable cells.

CD4+ and CD8+ cells can be modified with an expression vector encoding aCAR. In embodiments, these cells are then further sorted intosubpopulations of naïve, central memory and effector cells as describedabove by sorting for cell surface antigens unique to each of those cellpopulations. In addition, CD4+ or CD8+ cell populations may be selectedby their cytokine profile or proliferative activities. For example, CD4+T lymphocytes that have enhanced production of cytokines such as IL-2,IL-4, IL-10, TNFα, and IFNγ as compared to sham transduced cells ortransduced CD8+ cells when stimulated with antigen can be selected. Inother embodiments, naïve CD4+ T cells that have enhanced production ofIL-2 and/or TNFα are selected. Likewise, CD8+ cells that have enhancedIFNγ production are selected as compared to sham transduced CD8+ cells.

In embodiments, CD4+ and CD8+ cells that proliferate in response toantigen are selected. For example, CD4+ cells that proliferatevigorously when stimulated with antigen as compared to sham transducedcells, or CD8+ transduced cells are selected.

In some embodiments, CD4+ and CD8+ cells are selected that are cytotoxicfor antigen bearing cells. In embodiments, CD4+ are expected to beweakly cytotoxic as compared to CD8+ cells.

The disclosure contemplates that combinations of CD4+ and CD8+ T cellswill be utilized in the compositions. In one embodiment, combinations ofCAR transduced CD4+ cells can be combined with CD8+ antigen reactivecells to the same antigenic specificity as the CAR. In otherembodiments, CAR transduced CD8+ cells are combined with antigenreactive CD4+ cells. In yet another embodiment, CAR modified CD4+ andCD8+ cells are combined.

As described herein, the disclosure contemplates that CD4+ and CD8+cells can be further separated into subpopulations, such as naïve,central memory, and effector cell populations. As described herein, insome embodiments, naïve CD4+ cells are CD45RO−, CD45RA+, CD62L+ CD4+ Tcells. In some embodiments, central memory CD4+ cells are CD62L positiveand CD45RO positive. In some embodiments, effector CD4+ cells are CD62Lnegative and CD45RO positive. Each of these populations may beindependently modified with a CAR.

As described herein, in embodiments, memory T cells are present in bothCD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMCsare sorted into CD62L-CD8+ and CD62L+ CD8+ fractions after staining withanti-CD8 and anti-CD62L antibodies. In some embodiments, expression ofphenotypic markers of central memory T_(CM) include CD62L, CCR7, CD28,CD3, and CD127 and are negative for granzyme B. In some embodiments,central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In someembodiments, effector T_(E) cells are negative for CD62L, CCR7, CD28,and CD127, and positive for granzyme B and perforin. In someembodiments, naïve CD8+T lymphocytes are characterized by CD8+, CD62L+,CD45RO+, CCR7+, CD28+CD127+, and CD45RO+. Each of these populations maybe independently modified with a CAR.

Each of the subpopulations of CD4+ and CD8+ cells can be combined withone another. In a specific embodiment, modified naïve CD4+ cells arecombined with modified central memory CD8+ T cells to provide asynergistic cytotoxic effect on antigen bearing cells, such as tumorcells.

Methods

The disclosure provides methods of making adoptive immunotherapycompositions and uses or methods of using these compositions forperforming cellular immunotherapy in a subject having a disease ordisorder.

In embodiments, a method of manufacturing the compositions comprisesobtaining a modified naïve CD4+ T helper cell, wherein the modifiedhelper T lymphocyte cell preparation comprises CD4+ T cells that have achimeric antigen receptor comprising an extracellular antibody variabledomain specific for the antigen associated with the disease or disorderand an intracellular signaling domain.

In another embodiment, a method further comprises obtaining a modifiedCD8+ cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cellpreparation comprises CD8+ cells that have a chimeric antigen receptorcomprising an extracellular antibody variable domain specific for theantigen associated with the disease or disorder and an intracellularsignaling domain of a T cell receptor.

In another embodiment, a method comprises obtaining a modified CD8+cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cellpreparation comprises CD8+ T cells that have a chimeric antigen receptorcomprising an extracellular antibody variable domain specific for theantigen associated with the disease or disorder and an intracellularsignaling domain of a T cell receptor, and further comprising combiningthe modified CD8+ cytotoxic T cells with an antigen specific CD4+ helpercell lymphocyte cell preparation.

The preparation of the CD4+ and CD8+ cells that are modified with a CARhas been described above as well as in the examples. Antigen specific Tlymphocytes can be obtained from a patient having the disease ordisorder or can be prepared by in vitro stimulation of T lymphocytes inthe presence of antigen. Subpopulations of CD4+ and CD8+ T lymphocytescan also be isolated as described herein and combined in the methods ofmanufacturing.

The disclosure also provides methods of performing cellularimmunotherapy in a subject having a disease or disorder comprising:administering a composition of any one of claims 1-19. In otherembodiments, a method comprises administering to the subject agenetically modified cytotoxic T lymphocyte cell preparation thatprovides a cellular immune response, wherein the cytotoxic T lymphocytecell preparation comprises CD8+ T cells that have a chimeric antigenreceptor comprising an extracellular antibody variable domain specificfor an antigen associated with the disease or disorder and anintracellular signaling domain of a T cell or other receptors and agenetically modified helper T lymphocyte cell preparation that elicitsdirect tumor recognition and augments the genetically modified cytotoxicT lymphocyte cell preparations ability to mediate a cellular immuneresponse, wherein the helper T lymphocyte cell preparation comprisesCD4+ T cells that have a chimeric antigen receptor comprising anextracellular antibody variable domain specific for the antigenassociated with the disease or disorder and an intracellular signalingdomain of a T cell receptor.

In another embodiment, a method of performing cellular immunotherapy insubject having a disease or disorder comprises: administering to thesubject a genetically modified helper T lymphocyte cell preparation,wherein the modified helper T lymphocyte cell preparation comprises CD4+T cells that have a chimeric antigen receptor comprising a extracellularantibody variable domain specific for an antigen associated with thedisease or disorder and an intracellular signaling module of a T cellreceptor. In an embodiments, the method further comprises administeringto the subject a genetically modified cytotoxic T lymphocyte cellpreparation, wherein the modified cytotoxic T lymphocyte cellpreparation comprises CD8 positive cells that have a chimeric antigenreceptor comprising a extracellular antibody variable domain specificfor the antigen associated with the disease or disorder and anintracellular signaling module of a T cell receptor.

Another embodiment describes a method of performing cellularimmunotherapy in a subject having a disease or disorder comprising:analyzing a biological sample of the subject for the presence of anantigen associated with the disease or disorder and administering theadoptive immunotherapy compositions described herein, wherein thechimeric antigen receptor specifically binds to the antigen.

A CAR is produced that has a component that provides for specificbinding to an antigen associated with a disease or conditions, such as asolid tumor, cancer, viral infection, and an infection with a parasite.In embodiments, the intracellular signaling module of a T cell receptorof the chimeric antigen receptor comprises a transmembrane domain, aCD28 signaling domain, and a CD3 intracellular signaling domain, orother domains of T cell costimulatory molecules. In some embodiments,the intracellular signaling molecule comprises the CD3 intracellulardomain, a CD28 domain, a CD28 transmembrane and signaling domain linkedto a CD3 intracellular domain, or other domains of T cell costimulatorymolecules.

In alternative embodiments, the T cells can be modified with arecombinant T cell receptor. TCR could be specific for any antigen,pathogen or tumor. There are TCRs for many tumor antigens in melanoma(MART1, gp100 for example), leukemia (WT1, minor histocompatibilityantigens for example), breast cancer (her2, NY-BR1 for example).

In some embodiments, the CD4+ T helper lymphocyte cell is selected fromthe group consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells or bulk CD4+ T cells. In a specificembodiment, CD4+ helper lymphocyte cell is a naïve CD4+ T cell, whereinthe naïve CD4+ T cell comprises a CD45RO−, CD45RA+, CD62L+ CD4+ T cell.In yet other embodiments, the CD8+ T cytotoxic lymphocyte cell isselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells or bulk CD8+ T cells. In aspecific embodiment, the CD8+ cytotoxic T lymphocyte cell is a centralmemory T cell wherein the central memory T cell comprises a CD45RO+,CD62L+, CD8+ T cell. In a specific embodiment, the CD8+ cytotoxic Tlymphocyte cell is a central memory T cell and the CD4+ helper Tlymphocyte cell is a naïve CD4+ T cell.

In embodiments, the CD8+ T cell and the CD4+ T cell are both geneticallymodified with a CAR comprising an antibody heavy chain domain thatspecifically binds a pathogen or tumor-specific cell surface antigen. Inother embodiments, the intracellular signaling domain of the CD8cytotoxic T cells is the same as the intracellular signaling domain ofthe CD4 helper T cells. In yet other embodiments, the intracellularsignaling domain of the CD8 cytotoxic T cells is different than theintracellular signaling domain of the CD4 helper T cells.

Subjects that can be treated by the present invention are, in general,human and other primate subjects, such as monkeys and apes forveterinary medicine purposes. The subjects can be male or female and canbe any suitable age, including infant, juvenile, adolescent, adult, andgeriatric subjects.

The methods are useful in the treatment of, for example, solid tumor,hematologic malignancy, melanoma, or infection with a virus or otherpathogen. Infections with pathogens include HIV, HCV, HBV, CMV, andparasitic disease. In some embodiments, the antigen associated with thedisease or disorder is selected from the group consisting of orphantyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,mesothelin, CEA, and hepatitis B surface antigen.

Subjects that can be treated include subjects afflicted with cancer,including but not limited to colon, lung, liver, breast, prostate,ovarian, skin (including melanoma), bone, and brain cancer, etc. In someembodiments the tumor associated antigens are known, such as melanoma,breast cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma,prostate cancer, etc. (in these embodiments memory T cells can beisolated or engineered by introducing the T cell receptor genes). Inother embodiments the tumor associated proteins can be targeted withgenetically modified T cells expressing an engineered immunoreceptor.Examples include but are not limited to B cell lymphoma, breast cancer,prostate cancer, and leukemia.

Subjects that can be treated also include subjects afflicted with, or atrisk of developing, an infectious disease, including but not limited toviral, retroviral, bacterial, and protozoal infections, etc. Subjectsthat can be treated include immunodeficient patients afflicted with aviral infection, including but not limited to Cytomegalovirus (CMV),Epstein-Barr virus (EBV), adenovirus, BK polyomavirus infections intransplant patients, etc.

Cells prepared as described above can be utilized in methods andcompositions for adoptive immunotherapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure. See, e.g., US PatentApplication Publication No. 2003/0170238 to Gruenberg et al; see alsoU.S. Pat. No. 4,690,915 to Rosenberg.

In some embodiments, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a treatment-effective amount.Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with human serum albumin.

A treatment-effective amount of cells in the composition is at least 2cells (for example, 1 CD8+ central memory T cell and 1 CD4+ helper Tcell subset) or is more typically greater than 10² cells, and up to 10⁶,up to and including 10⁸ or 10⁹ cells and can be more than 10¹⁰ cells.The number of cells will depend upon the ultimate use for which thecomposition is intended as will the type of cells included therein. Forexample, if cells that are specific for a particular antigen aredesired, then the population will contain greater than 70%, generallygreater than 80%, 85% and 90-95% of such cells. For uses providedherein, the cells are generally in a volume of a liter or less, can be500 mls or less, even 250 mls or 100 mls or less. Hence the density ofthe desired cells is typically greater than 10⁶ cells/ml and generallyis greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. Theclinically relevant number of immune cells can be apportioned intomultiple infusions that cumulatively equal or exceed 10⁹, 10¹⁰ or 10¹¹cells.

In some embodiments, the lymphocytes of the invention may be used toconfer immunity to individuals. By “immunity” is meant a lessening ofone or more physical symptoms associated with a response to infection bya pathogen, or to a tumor, to which the lymphocyte response is directed.The amount of cells administered is usually in the range present innormal individuals with immunity to the pathogen. Thus, the cells areusually administered by infusion, with each infusion in a range of from2 cells, up to at least 10⁶ to 10¹⁰ cells/m², preferably in the range ofat least 10⁷ to 10⁹ cells/m². The clones may be administered by a singleinfusion, or by multiple infusions over a range of time. However, sincedifferent individuals are expected to vary in responsiveness, the typeand amount of cells infused, as well as the number of infusions and thetime range over which multiple infusions are given are determined by theattending physician, and can be determined by routine examination. Thegeneration of sufficient levels of T lymphocytes (including cytotoxic Tlymphocytes and/or helper T lymphocytes) is readily achievable using therapid expansion method of the present invention, as exemplified herein.See, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at column 17.

The present invention is illustrated further in the examples sot forthbelow.

EXPERIMENTAL Example 1—T Cell Transduction and Analysis of CARExpression

A ROR1-specific CAR can be expressed in human CD8+ T cells and confersspecific recognition of ROR1+ B-cell tumors and not mature normal Bcells. We constructed a ROR1-specific chimeric antigen receptor thatwhen expressed in T cells from healthy donors or CLL patients conferredspecific recognition of primary B-CLL and mantle cell lymphoma.

Materials and Methods Cell Lines

Epstein-Barr virus transformed B cells (EBV-LCL) were generated asdescribed (25). The tumor cell lines Jeko-1, and, BALL-1, were providedby Drs Oliver Press and Jerald Radich (Fred Hutchinson Cancer ResearchCenter). All cell lines were maintained in RPMI, 10% fetal calf serum,0.8 mM L-glutamine, and 1% penicillin-streptomycin (LCL medium). K562cells were obtained from the American Type Culture Collection.

Transfection of K562 Cells with ROR1

For polymerase chain reaction (PCR)-amplification of the ROR1-gene,total RNA was obtained from B-CLL cells (RNeasyPlusKit; QIAGEN) andreverse transcribed into cDNA with M-MLVReverse Transcriptase(Invitrogen). PCR was performed with specific primers (ROR1-F:5-XhoIAGAGGAGGAATGCACCGGCC-3 and ROR1-R:5-XhoI-CACAGAAGGTACTTGTTGCGATGT-3) using Herculase-II DNA Polymerase(Stratagene). The PCR product was cloned into the MIGR-1 retroviralvector (23) and the sequence verified. Effectene transfection reagent(QIAGEN) was used to transfect Platinum-A cells (Cell Biolabs) withMIGR-1/ROR1 and produce ROR1-encoding retrovirus. K562 cells wereretrovirally transduced by centrifugation at 2500 rpm for 60 minutes at32° C., expanded, and the ROR1-positive subset was sort-purified.

Real-Time Quantitative PCR

First-strand cDNA of B-CLL, normal resting and activated B cells, andEBV-LCL was prepared as described in the previous paragraph.First-strand cDNA from normal tissues (Human Tissue panels I/II, BloodFractions) was obtained from Clontech. Expression of ROR1 mRNA wasanalyzed in duplicate and normalized to GAPDH. Amplifications wereperformed on an ABI Prism 7900 (Applied Biosystems) in a 50 reactionconsisting of 25 μL Power SYBR Green PCR Master Mix (AppliedBiosystems), 2.5 ng of cDNA, and 300 nM gene-specific forward andreverse primers:

ROR1-F 5-AGCGTGCGATTCAAAGGATT-3, ROR1-R 5-GACTGGTGCCGACGATGACT-3,GAPDH-F 5-GAAGGTGAAGGTCGGAGTC-3, and GAPDH-R  5-GAAGATGGTGATGGGATTTC-3.

The cycle threshold (Ct) was determined using SDS software v2.2.2(Applied Biosystems) and the level of gene expression calculated usingthe comparative Ct method (2-(ΔΔCt)).

Vector Construction and Generation of Lentivirus

CD20-CAR (CD20R-epHIV7) and green fluorescent protein (GFP)-encodinglentiviral vectors (GFP-epHIV7) were described previously (24). TheROR1-CAR was encoded in the same vector. A mouse mAb (clone 2A2) thatdemonstrated specific binding to human ROR1 expressed on primary B-CLLand MCL tumor lines was generated, cloned, and characterized in aprevious study. A codon-optimized nucleotide sequence encoding a scFvcontaining the VL and VH chain of mAb 2A2 was synthesized (GENEART) andcloned into CD20R-epHIV7 using NheI and RsrII restriction sites toreplace the CD20-specific scFv. Lentivirus was produced in 293T cellsco-transfected with the lentiviral vector and the packaging vectorspCHGP-2, pCMVRev2, and pCMV-G using Effectene (Qiagen). Medium waschanged 16 hours after transfection and lentivirus collected after 48hours.

Lentiviral Transduction and Isolation of CAR-Transduced Tcell Clones

PBMC from healthy donors and B-CLL patients, and sort-purified CD8+CD45RO+ CD62L+ central memory T cells (T_(CM)) were activated withanti-CD3 mAb (30 ng/mL) (25), and transduced in lentiviral supernatantsupplemented with 1 μg/mL polybrene (Sigma-Aldrich) and 50 IU/mLrecombinant human interleukin-2 (IL-2) on day 2 and 3 after activationby centrifugation at 2500 rpm for 60 minutes at 32° C. T cells wereexpanded in RPMI containing 10% human serum, 2 mM L-glutamine, and 1%penicillin streptomycin (CTL medium) (25). After expansion, an aliquotof each transduced T-cell line was stained with biotin-conjugatedanti-EGFR (epithelial growth factor receptor) mAb, streptavidin-PE, andanti-CD8 mAb. EGFR+ CD8+ T cells were sort purified and cloned bylimiting dilution (0.5 cells/well) (25). ROR1-CAR transduced T cellswere identified by staining with biotinylated recombinant Fc-ROR1extracellular domain fusion protein and streptavidin-PE. RecombinantROR1-protein was produced in transiently transfected 293F cells(Invitrogen), purified as described (26), and biotinylated using theBiotinTag kit (Sigma). GFP-transduced CD8+ T cells were identified byflow cytometry, sort-purified, and cloned in similar fashion.

Chromium Release and Cytokine Secretion Assays

Target cells were labeled with ⁵¹Cr (PerkinElmer) overnight, washed andincubated in triplicate at 1-2×10³ cells/well with effector T cells atvarious effector to target (E:T) ratios. Supernatants were harvested forγ counting after a 4-hour incubation, and specific lysis was calculatedusing the standard formula (25).

Results

Transduced CD8+ T cells were sort-purified using a biotinylatedanti-EGFR mAb and streptavidin conjugated dyes. ROR1-CAR expression onthe surface of the sort-purified T cells was evaluated by staining thecells with a biotinylated recombinant Fc-ROR1 extracellular domainfusion protein that directly binds to the scFv of the ROR1-CAR, andcostaining with streptavidin-conjugates. Fc-ROR1-protein specificallystained CD8+ T cells transduced with the ROR1-CAR lentiviral vector butnot CD8+ T cells transduced with a control lentiviral vector encodingGFP (FIG. 1).

We established ROR1-CAR transduced (n=10) and control GFP-transducedCD8+ T-cell clones (n=4) by limiting dilution and confirmed the stablesurface expression of the CAR after multiple rounds of in vitroexpansion. There was no apparent difference in the growth of ROR1-CARtransduced compared with untransduced or GFP-transduced T-cell clones(data not shown).

The ROR1-CAR transduced T-cell clones efficiently lysed primary B-CLLand K562 cells that were stably transfected with the ROR1-gene, but notnative, ROR1-negative K562 cells, demonstrating specific recognition ofROR1 (FIG. 2).

Discussion

Adoptive immunotherapies that employ CAR-modified T cells are beinginvestigated in clinical trials for B-cell malignancies. The surfacemolecules that are being targeted are B-cell lineage-specific andinclude CD19, which is expressed on normal B-lineage cells from thepro-B-cell stage to plasma cells, and CD20, which is expressed on normalB cells from the pre-B-cell stage to memory B cells. Thus, ananticipated outcome of effective therapy targeting these molecules isdepletion of normal B cells and B-cell precursors. Gene expressionprofiling studies have identified genes that are preferentially orexclusively expressed by malignant but not by normal B cells and ROR1emerged as a CLL signature gene in 2 independent analyses (27,28).Specific antibodies to ROR1 developed in CLL patients after vaccinationwith autologous tumor cells that had been modified to express CD154 andtreatment with lenalidomide without apparent toxicity to normal tissues,suggesting this tumor antigen may be a suitable target for immunotherapy(29,30).

Our studies illustrate the potential to target ROR1-positive malignantcells with engineered T cells expressing a ROR1-CAR. CD8+ ROR1-CAR Tcells could be derived from both normal donors and CLL patients afterlentiviral transduction of either bulk PBMCs or sort-purified TCM, thatin animal models persist for extended periods after adoptive transfer(31). ROR1-CAR transduced T cells efficiently lysed primary B-CLL, butnot normal resting or activated B-cells. These T cells produced effectorcytokines including TNF-α, IFNγ, and IL-2, and were capable ofproliferating in response to ROR1-expressing tumor cells.

Example 2—Generation of CD4+ CAR T Cell Lines and Analysis of EffectorFunction

CD4+ ROR1-CAR T cells can be generated from PBMC of healthydonors/CLL-patients. A ROR1-specific CAR can be expressed in human CD4+T cells and confers specific recognition of ROR1+ B-cell tumors but notmature normal B cells.

Materials and Methods Cell Lines

Epstein-Barr virus transformed B cells (EBV-LCL) were generated asdescribed (25). The tumor cell lines Jeko-1, and BALL-1 were provided byDrs Oliver Press and Jerald Radich (Fred Hutchinson Cancer ResearchCenter). All cell lines were maintained in RPMI, 10% fetal calf serum,0.8 mM L-glutamine, and 1% penicillin-streptomycin (LCL medium). K562and 293T cells were obtained from the American Type Culture Collectionand cultured as directed.

Transfection of K562 Cells with ROR1

For polymerase chain reaction (PCR)-amplification of the ROR1-gene,total RNA was obtained from B-CLL cells (RNeasyPlusKit; QIAGEN) andreverse transcribed into cDNA with M-MLVReverse Transcriptase(Invitrogen). PCR was performed with specific primers (ROR1-F:5-XhoIAGAGGAGGAATGCACCGGCC-3 and ROR1-R:5-XhoI-CACAGAAGGTACTTGTTGCGATGT-3) using Herculase-II DNA Polymerase(Stratagene). The PCR product was cloned into the MIGR-1 retroviralvector (23), and sequence verified. Effectene transfection reagent(QIAGEN) was used to transfect Platinum-A cells (Cell Biolabs) withMIGR-1/ROR1 and produce ROR1-encoding retrovirus. K562 cells wereretrovirally transduced by centrifugation at 2500 rpm for 60 minutes at32° C., expanded, and the ROR1-positive subset was sort-purified.

Vector Construction and Generation of Lentivirus

CD20-CAR (CD20R-epHIV7) and green fluorescent protein (GFP)-encodinglentiviral vectors (GFP-epHIV7) were described previously (24). TheROR1-CAR was encoded in the same vector. A mouse mAb (clone 2A2) thatdemonstrated specific binding to human ROR1 expressed on primary B-CLLand MCL tumor lines was generated, cloned, and characterized in aprevious study. A codon-optimized nucleotide sequence encoding a scFvcontaining the VL and VH chain of mAb 2A2 was synthesized (GENEART) andcloned into CD20R-epHIV7 using NheI and RsrII restriction sites toreplace the CD20-specific scFv. Lentivirus was produced in 293T cellscotransfected with the lentiviral vector and the packaging vectorspCHGP-2, pCMVRev2, and pCMV-G using Effectene (Qiagen). Medium waschanged 16 hours after transfection and lentivirus collected after 48hours.

Lentiviral Transduction and Isolation of CD4+ ROR1-CAR T Cell Lines

CD4+ T cells were isolated from PBMC of healthy donors and activatedwith anti-CD3 mAb (30 ng/mL) (25), and transduced in lentiviralsupernatant supplemented with 1 μg/mL polybrene (Sigma-Aldrich) and 50IU/mL recombinant human interleukin-2 (IL-2) on day 2 and 3 afteractivation by centrifugation at 2500 rpm for 60 minutes at 32° C. Tcells were expanded in RPMI containing 10% human serum, 2 mML-glutamine, and 1% penicillin streptomycin (CTL medium).(25) Afterexpansion, an aliquot of each transduced T-cell line was stained withbiotin-conjugated anti-EGFR (epithelial growth factor receptor) mAb,streptavidin-PE, and anti-CD4 mAb. EGFR+ CD4+ T cells were sort purifiedand expanded. ROR1-CAR transduced T cells were identified by stainingwith biotinylated recombinant Fc-ROR1 extracellular domain fusionprotein and streptavidin-PE. Recombinant ROR1-protein was produced intransiently transfected 293 cells (Invitrogen), purified as described(26), and biotinylated using the BiotinTag kit (Sigma). GFP-transducedCD4+ T cells were identified by flow cytometry, sort-purified, andcloned in similar fashion.

Chromium Release and Cytokine Secretion Assays

Target cells were labeled with ⁵¹Cr (PerkinElmer) overnight, washed andincubated in triplicate at 1-2×10³ cells/well with effector T cells atvarious effector to target (E:T) ratios. Supernatants were harvested forγ counting after a 4-hour incubation, and specific lysis was calculatedusing the standard formula (25).

For analysis of cytokine secretion, target and effector cells wereplated in triplicate wells at an E/T ratio of 2:1, and interferon IFNγ,tumor necrosis factor (TNF-α), and IL-2 were measured by multiplexcytokine immunoassay (Luminex) in supernatant removed after a 24-hourincubation.

CFSE Proliferation Assay

T cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester(CFSE; Invitrogen), washed, and plated with stimulator cells at a ratioof 2:1 in CTL medium containing 10 U/mL recombinant human IL-2. After a72-hour incubation, cells were labeled with anti-CD4 mAb and propidiumiodide (PI) to exclude dead cells from analysis. Samples were analyzedby flow cytometry, and cell division of live CD4+ T cells assessed byCFSE dilution.

Co-Culture Assay

ROR1-CAR transduced CD4+ T cells and ROR1-CAR transduced CD8+ cytotoxicT lymphocytes were labeled with CFSE, and co-cultured at a 2:1, 1:1 and1:2 ratio. The co-cultures were then stimulated with K562/ROR1 cells andcontrol K562 cells and cell proliferation measured by CFSE dye dilutionassay after 5 days of incubation. For flow analysis, samples werestained with conjugated anti-CD8 and anti-CD4 mAb to distinguish CD8+and CD4+ subsets.

Results

Generation of CD4⁺ ROR1-CAR T cells from PBMC of healthy donors and CLLpatients We have shown that ROR1, an oncofetal tyrosine kinase receptor,is uniformly expressed on CLL and MCL, and developed a ROR1-CAR from ananti-ROR1 mAb that confers specific recognition of malignant, but notmature normal B cells when expressed in CD8⁺ T cells (32). Here, wegenerated CD4⁺ ROR1-CAR T cells to analyze direct tumor recognition andtheir ability to augment CD8⁺ ROR1-CAR CTL. CAR-modified CD4⁺ T cellscould be readily generated from bulk peripheral CD4⁺ T cells of healthydonors (n=4) and CLL patients (n=4) using a ROR1-CAR encoding lentiviralvector. In this vector, we encoded a truncated EGFR (epithelial growthfactor receptor, tEGFR) domain downstream of the ROR1-CAR and aself-cleavable 2A element, to serve both as transduction marker and forthe enrichment of transgene expressing T cells with anti-EGFR mAb (FIG.3). We determined the frequency of CAR-modified T cells on d12 after asingle transduction with ROR1-CAR encoding lentivirus (MOI=3) using thetEGFR marker and found consistently higher transduction efficiencies inCD4⁺ compared to CD8⁺ CAR T cell lines obtained from the sameindividuals. To confirm expression of the ROR1-CAR on the surface ofCD4⁺ T cells, we utilized biotinylated recombinant Fc-ROR1 extracellulardomain fusion protein that directly binds to the scFv of the ROR1-CARand specifically stained CD4⁺ T cells transduced with ROR1-CARlentivirus but not untransduced control CD4⁺ T cells (FIG. 3). Weenriched transgene expressing CD4⁺ T cells using the tEGFR marker andexpanded the CAR-positive T cell subset by stimulation with anti-CD3mAb. More than 3-log expansion of CD4⁺ CAR T cells could be achieved atthe end of a 14-day stimulation cycle, which is equivalent to theamplification observed in CD8⁺ CAR CTL. After expansion, we confirmedstable expression of the ROR1-CAR on the cell surface of CD4⁺ CAR Tcells (data not shown) and analyzed recognition of ROR1-positive tumorcells.

CD4⁺ ROR1-CAR T Cells Specifically Recognize ROR1-Positive Tumors

We analyzed the effector function of CD4⁺ ROR1-CAR T cells againstROR1-positive primary tumor cells and tumor cell lines. We analyzed theability of CD4⁺ CAR T cells to confer direct cytotoxicity by chromiumrelease assay (CRA) and detected weak but specific lysis ofROR1-positive target cells at the end of the standard 4-hour incubation(FIG. 4). We extended the CRA to 10 hours and observed a furtherincrease in specific lysis, however, the overall cytolytic activity ofCD4+ CAR T cells was still lower than CD8⁺ ROR1-CAR CTL (FIGS. 2 and 4).CD4⁺ ROR1-CAR T cells from both healthy donors and CLL patientsspecifically recognized primary CLL cells, the ROR1-positive tumor celllines Jeko-1 (MCL) and BALL-1 (B-ALL), and K562 cells that were stablytransfected with the ROR1-gene (K562/ROR1) but not native ROR1-negativeK562 cells by IFN-γ ELISA, demonstrating specific recognition of ROR1 onthe cell surface of target cells (FIG. 5A). Multiplex cytokine analysisrevealed production of other Th1 cytokines such as TNF-α and IL-2 atsignificantly higher levels compared to CD8⁺ CAR CTL, and production ofIL-4, IL-10 and IL-17 (FIG. 5B).

Next, we evaluated the proliferation of CD4⁺ CAR T cells afterstimulation with ROR1-positive tumor cells by CFSE staining and usedstringent culture conditions without addition of exogenous cytokines toremove any potential unspecific stimulus. CD4⁺ CAR T cells showeddramatic and specific proliferation in response to ROR1-positive tumorcells. Both the percentage of T cells that was induced to proliferateand the number of cell divisions that the proliferating subset performedwas significantly higher in CD4⁺ compared to CD8⁺ CAR T cells (FIG. 6).Collectively, our data demonstrate that CD4⁺ T cells obtained from bothhealthy donors and CLL patients acquire anti-tumor reactivity aftergenetic modification with a ROR1-specific CAR. Moreover, the ability toproliferate in the absence of exogenous cytokines and to produce highlevels of Th1 cytokines suggest that CD4⁺ CAR T cells exert typicalhelper functions after stimulation through the CAR and in addition toconferring direct anti-tumor effects, could also be utilized to augmentCD8⁺ CAR CTL.

CAR-Modified, but not Untransduced CD4⁺ T Cells Provide Help to CD8⁺ CARCTL

To analyze whether CD4⁺ CAR T cells are able to provide help to CD8⁺ CARCTL, we performed co-culture experiments with CAR-transduced and controluntransduced polyclonal CD4⁺ and CD8⁺ T cell lines that we establishedfrom healthy donors and CLL patients. As readout for provision of help,we defined an improvement in tumor-specific CD8⁺ effector function inthe presence of CD4⁺ T cells compared to CD8⁺ T cells cultured alone. Wecombined either CAR-transduced or untransduced control CD4⁺ T cells withCD8⁺ CAR CTL at distinct CD4:CD8 ratios (2:1, 1:1, 1:2), stimulated themwith ROR1-positive tumor cells and measured proliferation by CFSE dyedilution. We found, that the addition of CAR-transduced, but notuntransduced CD4⁺ T cells to CD8⁺ CAR CTL significantly increasedspecific proliferation of the CD8⁺ subset compared to CD8⁺ CAR CTL alone(FIG. 7). The increase in proliferation was most pronounced, when atleast an equivalent amount of CD4⁺ CART cells (CD4:CD8 ratio of 2:1 or1:1) was added to the co-culture. The combination of untransduced CD4⁺with untransduced CD8⁺ T cells served as additional control and did notinduce unspecific proliferation in the CD8⁺ subset (data not shown).

Discussion

Gene expression profiling studies have identified genes that arepreferentially or exclusively expressed by malignant but not by normal Bcells and ROR1 emerged as a CLL signature gene in 2 independent analyses(27,28). Our studies illustrate the potential to target ROR1-positivemalignant cells with engineered T cells expressing a ROR1-CAR. CD8 andCD4+ ROR1-CAR T cells could be derived from normal donors afterlentiviral transduction of either bulk PBMCs or sort-purified T cells.CD8+ ROR1-CAR transduced T cells efficiently lysed primary B-CLL, butnot normal resting or activated B-cells. CD4+ ROR1-CAR transduced Tcells weakly lysed primary B-CLL, but not normal resting or activatedB-cells. These T cells produced effector cytokines including TNF-α,IFNγ, IL-2, IL-4, and IL-10. CAR-transduced CD4+ T cells producedsignificantly higher amounts of cytokines than the transduced CD8+cells. Both cell types were capable of proliferating in response toROR1-expressing tumor cells. Again, CD4+ ROR1-CAR T cells proliferated2-3 fold higher than CD8+ ROR1-CAR CTLs. These results indicate that thetransduced CD4+ helper T cells exert typical helper functions suggestingthey could be utilized to augment CD8+ CAR CTLs.

Example 3—the Effector Function of CD4+ ROR1-CAR T Cells from Derivedfrom Naïve, Central and Effector Memory Subsets

The effector function of CD4 T cells derived from naïve, central andeffector memory subsets and then modified with the ROR1 CAR werecompared.

Materials and Methods Sort Purification of Naïve, Central, and EffectorMemory CD4 Cells

CD4+ T cells were isolated from PBMC of a healthy donor using negativemagnetic bead selection (Miltenyi CD4 isolation kit) that yieldsuntouched CD4+ T cells. The CD4+ fraction was labeled with conjugatedanti-CD45RA, anti-CD45RO and anti-CD62L mAb and flow sort purified usinga FACS Aria flow sorter (BD Biosciences), and naïve (CD45RA+ CD45RO−CD62L+), central memory (CD45RA− CD45RO+ CD62L+) and effector memory(CD45RA− CD45RO+ CD62L−) CD4+ T cells purified based on expression ofthese defined markers.

CFSE Proliferation Assay

T cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester(CFSE; Invitrogen), washed, and plated with stimulator cells at a ratioof 2:1 in CTL medium containing 10 U/mL recombinant human IL-2. After a72-hour incubation, cells were labeled with anti-CD8 or CD4 mAb andpropidium iodide (PI) to exclude dead cells from analysis. Samples wereanalyzed by flow cytometry, and cell division of live CD8+ and CD4+ Tcells assessed by CFSE dilution.

Cytokine Assays

For analyses of cytokine secretion, target and effector cells wereplated in triplicate wells at an E/T ratio of 2:1, and interferon INFγ,tumor necrosis factor (TNF-α), and IL-2 were measured by multiplexcytokine immunoassay (Luminex) in supernatant removed after a 24-hourincubation.

Results

We flow sort purified CD4⁺ N, central (CM) and effector memory (EM) CD4⁺T cells from the peripheral blood of 3 healthy donors based onexpression of CD45RA, CD45RO and CD62L (FIG. 8A), and compared theireffector function after modification with the ROR1-CAR. We achievedsimilarly high transduction efficiencies in CAR T cell lines derivedfrom each of the three subsets. Multiparameter flow cytometry afterenrichment of transgene expressing T cells showed expression of CD45ROand loss of CD45RA in the CD4⁺ N CAR T cell line, consistent with anactivated phenotype after the lentiviral transduction. The CD4⁺ N, CMand EM CAR T cell lines retained differential expression of CD62L,confirming that the initial flow sort purification had been performedwith high purity.

Then, we analyzed tumor recognition, cytokine secretion andproliferation of CD4⁺ CAR T cells derived from N, CM and EM subsets andcompared them to the CAR T cell lines generated from bulk CD4+ T cells.We observed specific recognition of ROR1-positive tumor cells by IFN-γELISA in each of the cell lines. Multiplex cytokine analysis revealedthat CD4⁺ CAR T cells derived from the N subset produced by far thehighest levels of Th1 cytokines, especially IL-2 (FIG. 8C) and CFSE dyedilution showed they proliferated most vigorously in response tostimulation with ROR1-positive tumor cells (FIG. 8B).

Discussion

Our studies illustrate the potential to target ROR1-positive malignantcells with engineered T cells expressing a ROR1-CAR. CD8 and CD4+ROR1-CAR T cells could be derived from both normal donors afterlentiviral transduction of either bulk PBMCs and sort-purified T cellsfrom defined naïve or memory T cell subsets. CD4+ naïve, central memory,and effector T cells produced effector cytokines including TNFα, IFNγ,IL-2, IL-4, and IL-10. CAR-transduced CD4+ cells derived from the naïvesubset produced significantly higher amounts of TNFα and IL-2 thancentral and effector memory derived CD4+ CAR T cells after signalingthrough the CAR. All CD4 cell types were capable of proliferating inresponse to ROR1/K562, however in the CAR-transduced CD4+ cells derivedfrom the naïve subset, the percentage of T cells that was induced toproliferate and the number of cell divisions that the proliferatingsubset underwent were significantly higher. Both cytokine profile andproliferative capacity indicate that naïve CD4+ ROR1-CAR T cells may bebest suited to augment CD8+ ROR1-CAR CTL.

Example 4—Naive CD4+ T Cells are Better Helpers than Memory CD4+ T Cells

Naïve, central memory, and effector transduced CD4+ T cells wereco-cultured with transduced CD8+ cytotoxic T lymphocytes and theproliferative response of the cells was measured in response tostimulation with K562/ROR1 cells.

Materials and Methods Co-Culture

Naïve, central and effector memory derived ROR1-CAR transduced CD4+ Tcells and ROR1-CAR transduced CD8+ cytotoxic T lymphocytes derived fromnaïve and central memory CD8+ T cells were labeled with CFSE, and CD4+and CD8+ CAR T cell lines co-cultured at a 1:1 ratio. The co-cultureswere then stimulated with K562/ROR1 cells and control K562 cells andcell proliferation was measured by CFSE dye dilution assay after 5 daysof incubation. For flow analysis, samples were stained with conjugatedanti-CD8 and anti-CD4 mAb to distinguish CD8+ and CD4+ subsets.

Results

CD4⁺ Naïve CAR T Cells have a Superior Ability to Augment the EffectorFunction of CD8⁺ CAR CTL

We compared the helper function of CD4⁺ N, CM and EM CAR T cell lines todetermine whether the favorable cytokine profile and proliferativepotential of CD4⁺N CAR T cells would also translate into the strongesthelper effect for CD8⁺ CAR CTL. Previous work has demonstrated thatthere are intrinsic differences between N, CM and EM CD8⁺ T cells thataffect their potential utility for adoptive immunotherapy. Our group hasrecently shown that CM but not EM derived CD8⁺ T cells are able topersist for extended periods after adoptive transfer which makes them apreferred subset of CD8⁺ T cells for immunotherapy (33,34). Other groupssuggested that CD8⁺ N T cells may also possess favorable traits for usein T cell therapy (35,36). Thus, we generated CD8⁺ CAR CTLs from sortpurified N and CM T cells to determine the optimal combination of CD8⁺and CD4⁺ CAR T cell subsets. Following lentiviral transduction andenrichment of CAR-transduced CD8⁺ T cells using the tEGFR marker, weconfirmed tumor-reactivity of the CD8⁺ N, and CM CAR CTLs (data notshown) and performed co-culture experiments with CD4⁺ CAR T cells asbefore. As anticipated, co-culture of CD8⁺ N and CM CAR CTL with CD4⁺ NCAR T cells resulted in significantly higher tumor-specificproliferation of the CD8⁺ subset compared to co-culture with CD4⁺ CM orEM CAR T cells, or the CD8⁺ CAR CTL alone (FIG. 9). Out of allcombinations, maximum proliferation of the CD8⁺ CAR CTL in response tostimulation with ROR1-positive tumor cells was observed after co-cultureof CD4⁺ N CAR T cells with CD8⁺ CM CAR CTL (FIG. 9). Collectively, ourdata demonstrate that there are intrinsic differences between N, CM andEM CD4⁺ T cells in their cytokine profile and proliferative potential,with higher production of IL-2 and superior proliferation in CD4⁺ N Tcells. Our data suggest that sort purified N, rather than CM, EM or bulkCD4⁺ T cells may be best suited to augment the effector function of CD8⁺CTL, and complement previous work in CD8⁺ T cells that CM derived CD8⁺ Tcells possess favorable characteristics for use in adoptiveimmunotherapy.

Discussion

Collectively, these data demonstrate that the adoptive transfer ofROR1-CAR modified CD4⁺ and CD8⁺ T cells confers potent anti-tumorresponses in an in vivo model of aggressive systemic lymphoma andprovide evidence for a beneficial and synergistic effect of CD4⁺ CAR Tcells on the anti-tumor efficacy of CD8⁺ CAR CTL. Our data illustratehow the analysis of cell-intrinsic qualities can inform the rationaldesign of cell products containing both tumor-specific CD8⁺ and CD4⁺ Tcells to improve outcomes of cancer immunotherapy.

Example 5—Mouse Tumor Model of Systemic Mantle Cell Lymphoma(NSG/Jeko-1-ffLuc)

We examined the effect of providing CD4 help on the anti-tumor efficacyof ROR1-CAR modified CD8⁺ CTL in an in vivo model of aggressive systemicmantle cell lymphoma.

Materials and Methods

Sub-lethally irradiated NOD/SCID/gamma^(−/−) (NSG) mice were engraftedvia tail vein injection with 5×10⁵ Jeko-1 cells that had been stablytransfected with firefly luciferase (Jeko-1/ffLuc) to enable assessmentof tumor burden and distribution using bioluminescence imaging. Weconfirmed the consistent engraftment (take rate=100%) and development ofrapidly progressive disseminated lymphoma in NSG mice under theseconditions. Following tumor engraftment, groups of 3 mice receivedeither CD8⁺ CAR CTLs (group 1), CD4⁺ CART cells (group 2), a combinationof CD8⁺ and CD4⁺ ROR1-CAR transduced T cells (group 3), untransducedcontrol T cells (group 4,5,6) via tail vein injection or no treatment(group 7). The total number of transferred T cells was 10×10⁶ in allcases. We obtained eye bleeds from the mice 2 days after adoptivetransfer and confirmed the presence of ROR1-CAR transduced oruntransduced T cells in the peripheral blood.

Results

On day 6 after T-cell transfer, we performed bioluminescence imaging toevaluate tumor burden. The strongest anti-tumor effect was observed inmice that received the combination of CD8⁺ and CD4⁺ ROR1-CAR T cells,with >2 log reduction in bioluminescence signal compared to the controlgroup (FIG. 10). We also observed a strong anti-tumor effect in micethat received either CD8⁺ or CD4⁺ ROR1-CAR modified T cells, with >1 logreduction in bioluminescence signal compared to controls (FIG. 10).Importantly, the reduction in tumor burden after administration of theCD8⁺/CD4⁺ CAR T cell combination was greater than that of the CD8⁺ CARCTL and CD4⁺ CAR T cell groups combined suggesting that CD4⁺ CAR T cellsand CD8⁺ CAR CTL were working synergistically.

Discussion

Collectively, these data demonstrate that the adoptive transfer ofROR1-CAR modified CD4⁺ and CD8⁺ T cells confers potent anti-tumorresponses in an in vivo model of aggressive systemic lymphoma andprovide evidence for a beneficial and synergistic effect of CD4⁺ CAR Tcells on the anti-tumor efficacy of CD8⁺ CAR CTL. Our data illustratehow the analysis of cell-intrinsic qualities can inform the rationaldesign of cell products containing both tumor-specific CD8⁺ and CD4⁺ Tcells to improve outcomes of cancer immunotherapy.

Example 6—CD19 CAR T Cells Exhibit the Same Synergy

We examined the effect of providing CD4 help on the anti-tumor efficacyof CD19 modified CD8⁺ CTL in coculture in vitro and in an in vivo modelof aggressive systemic mantle cell lymphoma.

Materials and Methods

CD19 CART cells can be prepared as described in US 2008/0131415, whichis hereby incorporated by reference.

Co-Culture Assay

CD19-CAR transduced CD4+ T cells and CD19-CAR transduced CD8+ cytotoxicT lymphocytes were labeled with CFSE, and co-cultured at a 2:1, 1:1 and1:2 ratio. The co-cultures were then stimulated with K562/ROR1 cells andcontrol K562 cells and cell proliferation measured by CFSE dye dilutionassay after 5 days of incubation. For flow analysis, samples werestained with conjugated anti-CD8 and anti-CD4 mAb to distinguish CD8+and CD4+ subsets.

In Vivo Model

Sublethally irradiated NOD/SCID/gamma^(−/−) (NSG) mice were engraftedvia tail vein injection with 5×10⁵ Jeko-1 cells that had been stablytransfected with firefly luciferase (Jeko-1/ffLuc) to enable assessmentof tumor burden and distribution using bioluminescence imaging. Weconfirmed the consistent engraftment (take rate=100%) and development ofrapidly progressive disseminated lymphoma in NSG mice under theseconditions. Following tumor engraftment, groups of 3 mice receivedeither CD8⁺ CD19 CAR CTLs (group 1), CD4⁺ CD 19 CART cells (group 2), acombination of CD8⁺ and CD4⁺ CD19CAR transduced T cells (group 3),untransduced control T cells (group 4,5,6) via tail vein injection or notreatment (group 7). The total number of transferred T cells was 10×10⁶in all cases. We obtained eye bleeds from the mice 2 days after adoptivetransfer.

Results

FIG. 10 shows the superior ability of CD4+ CAR T-cell lines derived fromthe naïve subset to augment tumor-specific proliferation of centralmemory-derived CD8+ CAR CTL in co-culture experiments with CD8+ CD19-CARCTLs and CD4+ CD19-CAR T-cell lines, stimulated with the CD19+ mantlecell lymphoma tumor line Jeko-1. Although, CD4+ CAR T-cell lines derivedfrom the central or effector memory subset augment tumor-specificproliferation of central memory-derived CD8+ CAR CTL to much lessextent.

FIG. 11 shows that CD8+ CAR T cells and CD4+ CAR T cells independentlyconfer direct anti-tumor efficacy in a lymphoma model in immunodeficientmice (NOD/SCID-Raji). Mice received either CD19-CAR transduced orcontrol mock-transduced CD8+ central memory-derived (FIG. 11A), orCD19-CAR transduced or control mock-transduced CD4+ naïve-derived Tcells (FIG. 11B).

FIG. 12 shows the augmentation and synergistic effect CD4+ ROR1-CARmodified T cells on the anti-tumor efficacy of CD8+ ROR1-CAR CTLs in amouse tumor model of systemic mantle cell lymphoma (NSG/Jeko-1-ffLuc).Anti-tumor efficacy of ROR1-CAR modified CD8+ and CD4+ T cells in amouse tumor model of systemic aggressive mantle cell lymphoma(NSG/Jeko-1) was enhanced as compared to either cell population alone orwhen compared to untransduced cells.

FIG. 13 shows synergy of CD8+ and CD4+ CD19-CAR T cells in a mouse modelof systemic lymphoma (NSG/Raji). Engraftment of the Raji tumor wasconfirmed by bioluminescence imaging on day 6 after tumor inoculation(before treatment) (treatment scheme shown in FIG. 13A, tumorengraftment by bioluminescence shown in FIG. 13B). Analysis of tumorburden using bioluminescence imaging showed complete eradication of theRaji tumors in the cohorts of mice treated with CD8+ CD19-CAR T cells,and in mice treated with the combined CD8+ and CD4+ CD19-CAR T-cellproduct (after treatment middle black and grey bars, FIG. 13B). The micewere then challenged with a second inoculum of Raji tumor cells and thefrequency of CD4+ and CD8+ CAR T cells in the peripheral blood, andtumor engraftment were analyzed. In mice treated with a combined CD8+and CD4+ CAR T-cell product, significantly higher levels CD8+ CAR Tcells after the tumor challenge (FIG. 13C, lower panels), and completerejection of the Raji inoculum (after tumor challenge right grey bar,FIG. 13B). In contrast, in mice that had received CD8+ CD19-CAR CTLalone, we did not detect an increase in CAR T cells after the tumorchallenge (FIG. 13C) and the Raji tumor cells were able to engraft(after tumor challenge right black bar, panel FIG. 13B).

Discussion

Collectively, these data demonstrate that transducing the cells withanother CAR construct, CD19, CD19-CAR modified CD4⁺ and CD8⁺ T cellsconfer potent anti-tumor responses in an in vivo model of aggressivesystemic lymphoma and provide evidence for a beneficial and synergisticeffect of CD4⁺ CAR T cells on the anti-tumor efficacy of CD8⁺ CAR CTL.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allreferences and documents referred to herein are hereby incorporated byreference.

REFERENCES

-   1. Cheever, M. A., et al., Specificity of adoptive    chemoimmunotherapy of established syngeneic tumors. J. Immunol. 125,    711-714 (1980).-   2. Pahl-Seibert, M.-F. et al. Highly protective in vivo function of    cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by    T-cell receptor-based cell sorting. J. Virol. 79, 5400-5413 (2005).-   3. Riddell, S R. et al. Restoration of viral immunity in    immunodeficient humans by the adoptive transfer of T cell clones.    Science 257, 238-241 (1992).-   4. Walter, E. A. et al. Reconstitution of cellular immunity against    cytomegalovirus in recipients of allogeneic bone marrow by transfer    of T-cell clones from the donor. N. Engl. J. Med. 333, 1038-1044    (1995).-   5. Rooney, C. M. et al. Infusion of cytotoxic T cells for the    prevention and treatment of Epstein-Barr virus-induced lymphoma in    allogeneic transplant recipients. Blood 92, 1549-1555 (1998).-   6. Dudley, M. E. et al. Cancer regression and autoimmunity in    patients after clonal repopulation with antitumor lymphocytes.    Science 298, 850-854 (2002)/7.-   Bollard, C. M. et al. Cytotoxic T lymphocyte therapy for    Epstein-Barr virus+ Hodgkin's disease. J. Exp. Med. 200, 1623-1633    (2004).-   8. Dudley, M. E. et al. Adoptive cell transfer therapy following    nonmyeloablative but lymphodepleting chemotherapy for the treatment    of patients with refractory metastatic melanoma. J. Clin. Oncol. 23,    2346-2357 (2005).-   9. Gattinoni, L., Powell Jr, D. J., Rosenberg, S. A., &    Restifo, N. P. Adoptive immunotherapy for cancer: building on    success. Nat. Rev. Immunol. 6, 383-393 (2006).-   10. Blattman, J. N. & Greenberg, P. D. Cancer Immunotherapy: A    treatment for the masses. Science 305, 200-205 (2004).-   11. Kessels, H. W. H. G. et al. Immunotherapy through TCR gene    transfer. Nat. Immuno!. 2, 957-961 (2001).-   12. Stanislawski, T. et al. Circumventing tolerance to a human    MDM2-derived tumor antigen by TCR gene transfer. Nat. Immunol. 2,    962-970 (2001).-   13. Brentjens, R. J. et al. Eradication of systemic B-cell tumors by    genetically targeted human T lymphocytes co-stimulated by CD80 and    interleukin-15. Nat. Med. 9, 279-286 (2003).-   14. Morgan, R. A. et al. Cancer regression in patients after    transfer of genetically engineered lymphocytes. Science advance    online publication Aug. 31, (2006). DOI: 10.1126/science.1129003-   15. Bleakley, M. & Riddell, S. R. Molecules and mechanisms of the    graft versus leukemia effect. Nat. Rev. Cancer 4, 371-380 (2004).-   16. Dudley, M. E. et al. Adoptive transfer of cloned    melanoma-reactive T lymphocytes for the treatment of patients with    metastatic melanoma. J. Immunother. 24, 363-373 (2001).-   17. Yee, C. et al. Adoptive T cell therapy using antigen-specific    CD8+ T cell clones for the treatment of patients with metastatic    melanoma: In vivo persistence, migration, and antitumor effect of    transferred cells. Proc. Natl. Acad. Sci. USA 99, 16168-16173    (2002).-   18. Sallusto, F. et al., Central memory and effector memory T cell    subsets: function, generation, and maintenance. Annu. Rev. Immunol.    22, 745-763 (2004).-   19. Butcher, E. C. & Picker, L. J. Lymphocyte homing and    homeostasis. Science 272, 60-66 (1996).-   21. Dudley, M. E. et al. A phase I study of nonmyeloablative    chemotherapy and adoptive transfer of autologous tumor    antigen-specific T lymphocytes in patients with metastatic    melanoma. J. Immunother. 25, 243-251 (2002).-   22. Gattinorti, L. et al. Acquisition of full effector function in    vitro paradoxically impairs the in vivo antitumor efficacy of    adoptively transferred CD8+ T cells. J. Clin. Invest. 115, 1616-1626    (2005).-   23. Schmitt T M, Ciofani M, Petrie H T, Zuniga-Plucker J C.    Maintenance of T cell specification and differentiation requires    recurrent notch receptor-ligand interactions. J Exp Med. 2004;    200(4):469-479.-   24. Wang J, Press O W, Lindgren C G, et al. Cellular immunotherapy    for follicular lymphoma using genetically modified CD20-specific    CD8+ cytotoxic T lymphocytes. Mol Ther. 2004; 9(4): 577-586.-   25. Riddell S R, Greenberg P D. The use of anti-CD3 and anti-CD28    monoclonal antibodies to clone and expand human antigen-specific T    cells. J Immunol Methods. 1990; 128(2):189-201.-   26. Baskar S, Kwong K Y, Hofer T, et al. Unique cell surface    expression of receptor tyrosine kinase ROR1 in human B-cell chronic    lymphocytic leukemia. Clin Cancer Res. 2008; 14(2):396-404.-   27. Klein U, Tu Y, Stolovitzky G A, et al. Gene expression profiling    of B cell chronic lymphocytic leukemia reveals a homogeneous    phenotype related to memory B cells. J Exp Med. 2001;    194(11):1625-1638.-   28. Rosenwald A, Alizadeh A A, Widhopf G, et al. Relation of gene    expression phenotype to immunoglobulin mutation genotype in B cell    chronic lymphocytic leukemia. J Exp Med. 2001;-   29. Fukuda T, Chen L, Endo T, et al. Antisera induced by infusions    of autologous Ad-CD154-leukemia B cells identify ROR1 as an    oncofetal antigen and receptor for Wnt5a. Proc Natl Acad Sci USA.    2008; 105(8):3047-3052.-   30. Lapalombella R, Andritsos L, Liu Q, et al. Lenalidomide    treatment promotes CD154 expression on CLL cells and enhances    production of antibodies by normal B cells through a    PI3-kinase-dependent pathway. Blood. 2010; 115(13):2619-2629.-   31. Berger C, Jensen M C, Lansdorp P M, Gough M, Elliott C, Riddell    S R. Adoptive transfer of effector CD8+ T cells derived from central    memory cells establishes persistent T cell memory in primates. J    Clin Invest. 2008; 118(1):294-305.

What is claimed is:
 1. An adoptive cellular immunotherapy compositioncomprising chimeric antigen receptor-modified CD4⁺ T lymphocytes andchimeric antigen receptor-modified CD8⁺ T lymphocytes, wherein: (a) thechimeric antigen receptor-modified CD4+ T lymphocytes in the compositionconsist of helper T lymphocytes that contain a chimeric antigen receptorthat specifically binds to an antigen, and (b) the chimeric antigenreceptor-modified CD8+ T lymphocytes in the composition consist of CD8+cytotoxic T lymphocytes that are derived from a central memory-enrichedCD8+ cell population and contain a chimeric antigen receptor thatspecifically binds to the antigen.
 2. The adoptive cellularimmunotherapy composition according to claim 1, wherein the antigen isassociated with a disease or disorder selected from a solid tumor, ahematologic malignancy, a melanoma, and an infection with a pathogen. 3.The adoptive cellular immunotherapy composition according to claim 1,wherein the antigen is selected from ROR1, tEGFR, Her2, L1-CAM, CD19,CD20, CD22, mesothelin, and CEA.
 4. The adoptive cellular immunotherapycomposition according to claim 1, wherein the antigen is a pathogenspecific cell surface antigen selected from an HIV antigen, an HCVantigen, an HBV antigen, a hepatitis B surface antigen, a CMV antigen,and a parasitic antigen.
 5. The adoptive cellular immunotherapycomposition according to claim 1, wherein the chimeric antigen receptorof (a) and/or (b) comprises an extracellular antibody variable domain orsingle-chain antibody fragment specific for an antigen associated with adisease or disorder, and an intracellular signaling module.
 6. Theadoptive cellular immunotherapy composition according to claim 5,wherein each of the intracellular signaling module of the chimericantigen receptor contained by the CD4+ T lymphocytes and theintracellular signaling module of the chimeric antigen receptorcontained by the CD8+ T lymphocytes, individually, comprise (a) a CD28costimulatory domain and a CD3 intracellular signaling domain, or (b) a4-1BB costimulatory domain and a CD3 intracellular signaling domain. 7.The adoptive cellular immunotherapy composition according to claim 1,wherein (a) the intracellular signaling domain of the chimeric antigenreceptor contained by the CD8+ T lymphocytes is the same as theintracellular signaling domain of the chimeric antigen receptorcontained by the CD4+ T lymphocytes, or (b) the chimeric antigenreceptor contained by the CD8+ T lymphocytes is the same as the chimericantigen receptor contained by the CD4+ T lymphocytes.
 8. The adoptivecellular immunotherapy composition according to claim 1, wherein (a) theintracellular signaling domain of the chimeric antigen receptor in theCD8+ T lymphocytes is different from the intracellular signaling domainof the chimeric antigen receptor in the CD4+ T lymphocytes; or (b) thechimeric antigen receptor contained by the CD8+ T lymphocytes isdifferent than the chimeric antigen receptor contained by the CD4+ Tlymphocytes.
 9. The adoptive cellular immunotherapy compositionaccording to claim 1, wherein (a) at least 60% of the chimeric antigenreceptor-modified CD4+ T lymphocytes are surface positive for CD62L andCD45RA or CD45RO; or (b) at least 80% of the chimeric antigenreceptor-modified CD4+ T lymphocytes are surface positive for CD62L andCD45RA or CD45RO.
 10. The adoptive cellular immunotherapy compositionaccording to claim 1, wherein the CD4+ helper T lymphocytes are derivedfrom (a) a CD45RA+ CD62L+ naïve T cell-enriched CD4+ population; (b) aCD45RO+ CD62L+ central memory T cell-enriched CD4+ population; (c) aCD45RA+ CD62L+ naïve T cell-enriched and CD45RO+ CD62L+ central memory Tcell-enriched CD4+ population; or (d) a bulk CD4+ T cell population. 11.A method of treating a subject having cancer or an infectious disease,comprising administering the adoptive cellular immunotherapy compositionof claim
 1. 12. The method according to claim 11, wherein the cancer isselected from a solid tumor, a hematologic malignancy, or a melanoma.13. The method according to claim 11, wherein the infectious disease isa viral infection.
 14. The method according to claim 13, wherein theviral infection is selected from an infection with a hepatitis virus, aherpes virus, a retrovirus, or a flavivirus.
 15. The method according toclaim 11, wherein the chimeric antigen receptor-modified CD4⁺ Tlymphocytes are capable of augmenting the effector function of thechimeric antigen receptor-modified CD8⁺ cytotoxic T lymphocytes.
 16. Themethod according to claim 11, wherein at least 60% of the chimericantigen receptor-modified CD8+ cytotoxic T lymphocytes are derived froma central memory-enriched CD8+ T cell population.
 17. The methodaccording to claim 11, wherein at least 80% of the chimeric antigenreceptor-modified CD8+ cytotoxic T lymphocytes are derived from acentral memory-enriched CD8+ T cell population.
 18. A method ofmanufacturing the adoptive cellular immunotherapy composition of claim1, comprising: (a) expanding in vitro, individually, (i) a population ofcentral memory-enriched CD8⁺ T lymphocytes and (ii) a population of CD4⁺T lymphocytes, thereby generating an expanded CD8+ T lymphocytepopulation and an expanded CD4+ T lymphocyte population; (b) modifyingcells of the expanded CD8⁺ T lymphocytes by introducing a nucleic acidmolecule encoding a chimeric antigen receptor that specifically binds tothe antigen, and modifying cells of the expanded CD4+ T lymphocytes byintroducing a nucleic acid molecule encoding the chimeric antigenreceptor that specifically binds to the antigen; and (c) optionallymixing or combining cells of the modified CD8⁺ T lymphocytes and themodified CD4⁺ T lymphocytes; thereby generating the adoptive cellularimmunotherapy composition.